WO2017081876A1 - White mildew resistance marker for melon plants, white mildew-resistant melon plant, and method for producing white mildew-resistant melon plant using said white mildew-resistant melon plant - Google Patents

White mildew resistance marker for melon plants, white mildew-resistant melon plant, and method for producing white mildew-resistant melon plant using said white mildew-resistant melon plant Download PDF

Info

Publication number
WO2017081876A1
WO2017081876A1 PCT/JP2016/066009 JP2016066009W WO2017081876A1 WO 2017081876 A1 WO2017081876 A1 WO 2017081876A1 JP 2016066009 W JP2016066009 W JP 2016066009W WO 2017081876 A1 WO2017081876 A1 WO 2017081876A1
Authority
WO
WIPO (PCT)
Prior art keywords
powdery mildew
chromosome
melon plant
seq
polynucleotide
Prior art date
Application number
PCT/JP2016/066009
Other languages
French (fr)
Japanese (ja)
Inventor
一夫 小杉
龍平 有本
前田　大輔
彰人 加野
龍二 池末
Original Assignee
タキイ種苗株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by タキイ種苗株式会社 filed Critical タキイ種苗株式会社
Priority to TR2018/06450T priority Critical patent/TR201806450T1/en
Priority to ES201890033A priority patent/ES2691593B1/en
Priority to US15/775,637 priority patent/US10772273B2/en
Publication of WO2017081876A1 publication Critical patent/WO2017081876A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for fungal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
    • A01H6/344—Cucumis melo [melon]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the present invention relates to a powdery mildew resistance marker for melon plants, a powdery mildew resistant melon plant, and a method for producing a powdery mildew resistant melon plant using the same.
  • Non-Patent Document 1 powdery mildew fungus capable of infecting melon plants containing these resistance genes has emerged and has become a problem.
  • the present invention provides a powdery mildew resistance marker for a new melon plant, a powdery mildew resistant melon plant containing a powdery mildew resistance locus, and a method for producing a powdery mildew resistant melon plant using the same.
  • the purpose is to provide.
  • the powdery mildew resistance marker of the melon plant of the present invention comprises a powdery mildew resistance locus on chromosome 6 in a homozygous form,
  • the powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
  • Condition (1) The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1, The length of the amplified fragment is 133 bases or more.
  • Primer set 1 Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
  • Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  • the powdery mildew resistant melon plant of the present invention comprises a powdery mildew resistant locus on chromosome 6 in a homozygous form,
  • the powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
  • Condition (1) The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1, The length of the amplified fragment is 133 bases or more.
  • Primer set 1 Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
  • Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  • the method for producing a powdery mildew resistant melon plant of the present invention includes the following steps (a) and (b).
  • the present inventors have found a novel powdery mildew resistance locus as a powdery mildew resistance marker showing powdery mildew resistance in melon plants. Moreover, the melon plant containing the said powdery mildew resistance marker shows powdery mildew resistance. Therefore, according to the powdery mildew resistance marker of the melon plant of the present invention, for example, powdery mildew resistant melon plants can be easily screened. Moreover, since the powdery mildew resistant melon plant of this invention contains the said powdery mildew resistant gene locus, for example, it can show powdery mildew resistant, for example.
  • the powdery mildew resistance locus can impart powdery mildew resistance with a single locus (single factor), for example.
  • the powdery mildew resistant melon plant of this invention can obtain easily the progeny which shows powdery mildew resistance from F1 obtained by crossing with another melon plant, or its progeny, for example.
  • the melon plant containing the powdery mildew resistance marker also exhibits resistance to powdery mildew bacteria that can infect melon plants containing the powdery mildew resistance gene of the prior art document, for example. For this reason, since the powdery mildew resistant melon plant of this invention does not need the control by the conventional agrochemical, the problem of the labor and cost of the said agrochemical spraying can also be avoided, for example.
  • FIG. 1 is a schematic diagram showing relative seating positions of SNP (single nucleotide polymorphism) and the like on the sixth chromosome.
  • FIG. 2 is a photograph showing evaluation criteria for the disease index of melon plants in Example 1.
  • Powdery mildew resistance marker of melon plant The powdery mildew resistance marker of the melon plant of the present invention (hereinafter also referred to as “resistance marker”) is powdery mildew resistance on chromosome 6 as described above.
  • a locus (hereinafter also referred to as “resistance locus”) in a homozygous form, wherein the powdery mildew resistance locus on chromosome 6 is at least one of the following conditions (1) and (2): It satisfies the conditions.
  • the powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1, The length of the amplified fragment is 133 bases or more.
  • Primer set 1 Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
  • Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  • the powdery mildew resistance marker of the present invention comprises a powdery mildew resistance locus on chromosome 6 in a homozygous form, and the powdery mildew resistance locus on chromosome 6 is the condition (1). And (2) satisfying at least one of the conditions, and other configurations and conditions are not particularly limited.
  • the “melon plant” is a plant classified as Cucumis melo of the genus Cucumis .
  • examples of the powdery mildew pathogen include Sphaerotheca fuliginea (also referred to as “ Podosphaera xanthii ”), Erysiphe polygoni, and the like.
  • “powder resistance” is also referred to as “powder resistance”, for example.
  • the resistance means, for example, the ability to inhibit or suppress the occurrence and progression of disease caused by infection with the powdery mildew pathogen, specifically, for example, the occurrence of no disease, stop of progression of the disease that has occurred, and Any of suppression of the progression of the disease that has occurred (also referred to as “inhibition”) may be used.
  • the “chromosome” can also be referred to as a “linkage group”, for example. Therefore, in the present invention, the first to twelfth chromosomes of the melon plant can also be referred to as, for example, the first to twelfth linkage groups, and the sixth chromosome can also be referred to as, for example, the sixth linkage group.
  • the said linkage group can be determined with reference to the following reference 1 based on the base sequence information of the genome of a melon plant, for example.
  • the base sequence information of the melon plant genome can be obtained from, for example, the MELONOMICS Project website (https://melonomics.net/).
  • the 6th chromosome (sixth linkage group) in the present invention is based on, for example, the base sequence information Melon_genome_v3.5.1 of the melon plant published on the website of the Melonomics Project. Can be determined.
  • the resistance marker of the present invention contains the resistance locus on the sixth chromosome in a homozygous form, but the powdery mildew resistant melon plant is, for example, replaced with the sixth chromosome,
  • the resistance locus on the sixth chromosome may be included on any chromosome other than the sixth chromosome. That is, the powdery mildew resistant melon plant has chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 7, chromosome 8, chromosome 9, chromosome 10,
  • the resistance locus on the sixth chromosome may be included on either the chromosome or the twelfth chromosome.
  • the powdery mildew resistant melon plant includes the resistance marker on a chromosome other than the sixth chromosome, for example, one resistance locus may be included on a chromosome other than the sixth chromosome, or two resistances may be included. Sex loci may be included on chromosomes other than chromosome 6. In the latter case, the powdery mildew resistant melon plant may include, for example, the two resistance loci on the same chromosome or different chromosomes.
  • the powdery mildew resistance locus means a quantitative trait locus or gene region that provides powdery mildew resistance.
  • the quantitative trait locus (Quantitative Traits Loci; QTL) generally means a chromosomal region involved in the expression of quantitative traits.
  • QTL can be defined using molecular markers that indicate specific loci on the chromosome. Techniques for defining QTL using the molecular markers are well known in the art.
  • the molecular marker used for the regulation (hereinafter also referred to as “specific”) of the powdery mildew resistance locus is not particularly limited.
  • the molecular markers include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP (restriction fragment length polymorphism) markers, microsatellite markers, SCAR (sequence-characterized amplified region) markers, and CAPS. (Cleaved amplified polymorphic sequence) markers and the like.
  • the microsatellite marker include a STR (short tandem repeat) marker, a SSR (simple sequence repeat) marker, and the like.
  • the resistance gene locus may be specified by, for example, one type of marker, or may be specified by two or more types of markers.
  • the SNP marker for example, one SNP may be used as the SNP marker, or a combination of two or more SNPs may be used as the SNP marker.
  • the resistance locus satisfies the condition based on at least one of the combination of (i) the length of the amplified fragment amplified by the primer set and the base polymorphism (hereinafter also referred to as “SNP”). . That is, as described above, the resistance locus satisfies at least one of the conditions (1) and (2).
  • the resistance locus may satisfy, for example, (ii) a condition based on at least one of a base sequence of an amplified fragment amplified by a primer set and a base sequence including a combination of SNPs, as described later.
  • the conditions based on the base sequence of the amplified fragment amplified with the primer set and the base sequence of the region containing at least one of the base sequences including the combination of SNP may be satisfied, or the conditions of these combinations must be satisfied Also good.
  • the combination is not particularly limited, and examples thereof include the following combinations.
  • the resistance locus satisfies the above (i), but the present invention is not limited to this.
  • the condition combining (ii) and (iii) may be satisfied.
  • each SNP in each condition is a SNP newly identified by the present inventors, and if it is a person skilled in the art, the SNP sitting position based on a base sequence containing these SNPs described later. Can be identified.
  • the resistance locus is specified by the length of the amplified fragment amplified by the following primer set 1.
  • the length of the amplified fragment can be measured, for example, by performing gene amplification on a melon plant using the following primer set 1 and analyzing the obtained amplified fragment.
  • the analysis of the amplified fragment may be, for example, sequencing of the amplified fragment or analysis by electrophoresis or the like.
  • Primer set 1 below amplifies, for example, nucleotide sequences 5857028 to 5857159 on chromosome 6 in the above-mentioned genome sequence information of the melon plant.
  • Primer set 1 Forward primer 1 5'-AATCTCAACAAGTGAGCTTTTATTGT-3 '(SEQ ID NO: 1)
  • Reverse primer 1 5'-CATGATTATCTTCAATTTTCTTTTTGTC-3 '(SEQ ID NO: 2)
  • the length of the amplified fragment may be 133 bases or more, and the upper limit is not particularly limited.
  • the upper limit of the length of the amplified fragment is, for example, 140 bases or less and 135 bases or less, and the range thereof is, for example, 133 to 140 bases length, 133 to 135 bases length.
  • the length of the amplified fragment is preferably 133 bases long. The relationship between the length of the amplified fragment amplified with the primer set 1 and the powdery mildew resistance has not been reported so far, and the powdery mildew resistance first discovered by the present inventors. Is the length of the new amplified fragment involved in
  • the resistance locus is specified by a combination of nine types of SNPs in the nucleotide sequence of SEQ ID NO: 3 (hereinafter also referred to as “first SNP set”).
  • the nine types of SNPs are 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th SNPs in the base sequence of SEQ ID NO: 3 below.
  • the 45th , 48th , 49th , 51st , 108th , 120th, 139th, 214th, and 327th SNPs are respectively referred to as “SNP1 45 ”, “SNP1 48 ”, “SNP1 49 ”, “SNP1 51 ”, “SNP1 108 ”. ”,“ SNP1 120 ”,“ SNP1 139 ”,“ SNP1 214 ”, and“ SNP1 327 ”.
  • the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , the SNP1 120 , the SNP1 139 , the SNP1 214 , and the SNP1 327 are parentheses in the base sequence of the following SEQ ID NO: 3, respectively.
  • the base sequence of SEQ ID NO: 3 below can be obtained from, for example, a melon plant deposited under the accession number FERM BP-22291 described later.
  • the base sequence of SEQ ID NO: 3 below is, for example, the base sequence corresponding to the 5586975th to 587541st base sequence on chromosome 6 in the above-mentioned base sequence information of the melon plant genome.
  • the 1st to 8th and 9th bases are respectively A, A, T, T, T, A, A, T And polymorphisms that are C.
  • the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , the SNP1 120 , the SNP1 139 , the SNP1 214 , and the SNP1 327 in the first SNP set are respectively A , A, T, T, T, A, A, T, and C
  • the melon plant is resistant to powdery mildew and in the case of a combination of bases other than the above-mentioned combinations of bases (for example, A, T, In the case of T, T, T, T, A, G, and T, in the case of G, A, A, A, G, C, A, C, and T)
  • the melon plant is susceptible to powdery mildew. Show.
  • the relationship between the first SNP set and powdery mildew resistance has not been reported so far, and a novel SNP related to powdery mildew resistance, which was first discovered by the present inventors, was found. It is a
  • the resistance locus is further specified by, for example, the 204th to 212th and 213th base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in the base sequence of SEQ ID NO: 3. May be.
  • the base sequence of SEQ ID NO: 7 is a base sequence surrounded by a square in the base sequence of SEQ ID NO: 3.
  • the first SNP set includes a combination of bases exhibiting resistance to powdery mildew and the base sequence corresponding to the base sequence of SEQ ID NO: 3 includes the base sequence of SEQ ID NO: 7,
  • the plant is resistant to powdery mildew, and when it does not contain the nucleotide sequence of SEQ ID NO: 7, it indicates that the melon plant is susceptible to powdery mildew.
  • the relationship between the nucleotide sequence of SEQ ID NO: 7 and powdery mildew resistance has not been reported so far, and was first discovered by the present inventors and related to the powdery mildew resistance. Is the base sequence.
  • the resistance locus may satisfy, for example, one of the conditions (1) and (2), or may satisfy both conditions, and correlation with powdery mildew resistance. Therefore, it is preferable to satisfy both conditions.
  • the resistance locus may further satisfy at least one of the following conditions (3) and (4) as a condition based on the combination of the SNPs, for example.
  • the powdery mildew resistance locus on the sixth chromosome is specified by polymorphisms of the 50th, 141st and 266th bases in the base sequence of SEQ ID NO: 4.
  • the powdery mildew resistance locus on the sixth chromosome is identified by polymorphisms of the 99th, 174th, 184th, 199th and 200th bases in the base sequence of SEQ ID NO: 5.
  • the resistance locus is specified by, for example, a combination of three types of SNPs in the base sequence of SEQ ID NO: 4 (hereinafter also referred to as “second SNP set”).
  • the three types of SNPs are, for example, the 50th, 141st, and 266th SNPs in the base sequence of SEQ ID NO: 4.
  • the 50th, 141st, and 266th SNPs are also referred to as “SNP2 50 ”, “SNP2 141 ”, and “SNP2 266 ”, respectively.
  • the SNP 2 50 , the SNP 2 141 , and the SNP 2 266 are the first, second, and third bases in the underlined portion in parentheses in the base sequence of SEQ ID NO: 4, respectively.
  • the base sequence of SEQ ID NO: 4 can be obtained, for example, from a melon plant deposited under accession number FERM BP-22291 described later.
  • the base sequence of SEQ ID NO: 4 is, for example, a base sequence corresponding to the 6398131 to 6398627th base sequence on chromosome 6 in the genome sequence information of the melon plant described above.
  • the second SNP set indicates, for example, polymorphisms in which the first, second, and third bases are C, A, and T, respectively, in the underlined bases surrounded by the brackets of SEQ ID NO: 4. That is, for example, when the SNP2 50 , the SNP2 141 , and the SNP2 266 in the second SNP set are C, A, and T, respectively, the melon plant is resistant to powdery mildew, and the combination of the bases For combinations of bases other than (eg, for T, T, and A), the melon plant is susceptible to powdery mildew.
  • the relationship between the second SNP set and powdery mildew resistance has not been reported so far, and a novel SNP related to powdery mildew resistance, which was first discovered by the present inventors, was found. It is a combination.
  • the resistance locus may be further specified, for example, by a combination of the 132nd and 457th SNPs in the base sequence of SEQ ID NO: 4.
  • the 132nd and 457th SNPs are also referred to as “SNP2 132 ” and “SNP2 457 ”, respectively.
  • the SNP2 132 and the SNP2 457 are the first and second bases surrounded by a square in the base sequence of SEQ ID NO: 4, respectively.
  • the SNP2 132 and the SNP2 457 represent polymorphisms in which the first and second bases are T and A, respectively, in the underlined base surrounded by the square of the SEQ ID NO: 4. That is, for example, when the above-described second SNP set includes a combination of bases exhibiting powdery mildew resistance, and the SNP2 132 and the SNP2 457 are T and A, respectively, the melon plant has the powdery mildew resistance. In the case of a combination of bases other than the combination of bases, it indicates that the melon plant is susceptible to powdery mildew. In addition, the relationship between the SNP2 132 and the SNP2 457 and powdery mildew resistance has not been reported so far, and was first discovered by the present inventors and related to the powdery mildew resistance. Of SNPs.
  • the resistance locus is specified by, for example, a combination of five types of SNPs in the base sequence of SEQ ID NO: 5 (hereinafter also referred to as “third SNP set”).
  • the five types of SNPs are, for example, the 99th, 174th, 184th, 199th and 200th SNPs in the base sequence of SEQ ID NO: 5.
  • the 99th, 174 , 184 , 199 , and 200th SNPs are also referred to as “SNP3 99 ”, “SNP3 174 ”, “SNP3 184 ”, “SNP3 199 ”, and “SNP3 200 ”, respectively.
  • the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 are the 1st to 4th and 5th bases in the underlined part in the base sequence of the SEQ ID NO: 5, respectively.
  • the base sequence of SEQ ID NO: 5 can be obtained, for example, from a melon plant deposited under accession number FERM BP-22291 described later.
  • the base sequence of SEQ ID NO: 5 is, for example, the base sequence corresponding to the 5269642-5270103 base sequence on chromosome 6 in the genome sequence information of the melon plant described above.
  • the third SNP set includes, for example, polymorphisms in which the first to fourth and fifth bases are C, G, T, A, and T, respectively, in the underlined bases enclosed in parentheses of SEQ ID NO: 5. Show. That is, for example, when the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 in the third SNP set are C, G, T, A, and T, respectively, In the case of a combination of bases other than the above-mentioned base combinations that are resistant to powdery mildew (for example, in the case of T, A, C, T, and C), it indicates that the melon plant is susceptible to powdery mildew. . In addition, the relationship between the third SNP set and powdery mildew resistance has not been reported so far, and a novel SNP related to powdery mildew resistance, which was first discovered by the present inventors, was found. It is a combination.
  • the combination of the conditions satisfied by the resistance locus is not particularly limited.
  • the following combinations It can be illustrated.
  • the resistance locus satisfies at least one of the conditions (1) and (2), but the resistance locus is not limited thereto, and the conditions (1) and ( Instead of at least one of the conditions of 2), at least one of the conditions (3) and (4) may be satisfied.
  • the resistance locus may satisfy one of the conditions (3) and (4), or may satisfy both conditions, and has a correlation with powdery mildew resistance. Since it is higher, it is preferable to satisfy both conditions.
  • the resistance locus is based on, for example, at least one of the nucleotide sequence of an amplified fragment amplified by a primer set and a nucleotide sequence including a combination of SNPs.
  • the condition may be satisfied.
  • at least one of the following conditions (5) and (6) may be satisfied.
  • the following condition (5) the following (a2) and (a3) are polynucleotides having a function equivalent to that of the following (a1) regarding the powdery mildew resistance at the resistance locus.
  • the following (b2) and (b3) are polynucleotides having functions equivalent to those in the following (b1) with respect to the powdery mildew resistance at the resistance locus.
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (a) below.
  • polynucleotide (a2) comprising the nucleotide sequence of SEQ ID NO: 6 (a1), polynucleotide (a1) below (a1), (a2), or (a3): one or several bases deleted in (a1)
  • a polynucleotide comprising a base sequence that has been substituted, inserted and / or added (a3) a polynucleotide comprising a base sequence having 80% or more identity to the base sequence of (a1) above
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (b) below.
  • B Polynucleotide (b2) of the following (b1), (b2), or (b3) (b1) SEQ ID NO: 3 (b2) 45th base (A), 48th of (b1) Base (A), 49th base (T), 51st base (T), 108th base (T), 120th base (A), 139th base (A), 214th base (T) and a polynucleotide comprising a base sequence in which the 327th base (C) is conserved and one or several bases are deleted, substituted, inserted and / or added in a base sequence other than the base ( b3) 45th base (A), 48th base (A), 49th base (T), 51st base (T), 108th base (T), 120th base of (b1) Base (A), 139th base (A) , 214th base (T), and 327th base (b
  • the base sequence of SEQ ID NO: 6 is as follows.
  • the polynucleotide (a1) (base sequence of SEQ ID NO: 6) can be obtained, for example, by performing gene amplification on, for example, a melon plant using the primer set 1.
  • the base sequence of SEQ ID NO: 6 is, for example, the base sequence of the amplified fragment containing the forward primer 1 in the amplified fragment.
  • the polynucleotide (a1) can be obtained, for example, from a melon plant deposited under the deposit number FERM BP-22291 described later.
  • SEQ ID NO: 6 5'-AATCTCAACAAGTGAGCTTTTATTGTAAAAAATACAACACAAGTAAGAGTGTGTGTATTTATAATTGAAAGAAGAAGAAGAAGAAGAAGAAGAAAACAAGACAAAAAGAAAATTGAAGATAATCATG-3 '
  • the “one or several” is, for example, 1 to 27, 1 to 20, 1 to 15, 1 to 7, 1 to 5, 1 to 4, One to three, one or two.
  • the numerical range of the number of bases and the like discloses, for example, all positive integers belonging to the range. That is, for example, the description “1 to 5” means all disclosures of “1, 2, 3, 4, 5” (hereinafter the same).
  • the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the “identity” can be determined by aligning two base sequences (hereinafter the same).
  • the underlined first to eighth and ninth bases surrounded by parentheses in SEQ ID NO: 3 are the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , It is a base corresponding to the polymorphisms of SNP1 120 , SNP1 139 , SNP1 214 , and SNP1 327 .
  • the polynucleotide (b1) can be obtained, for example, from a melon plant deposited under the accession number FERM BP-22291 described later.
  • the “one or several” is, for example, 1 to 112, 1 to 84, 1 to 62, 1 to 56, 1 to 28, 1 to 23, 1 to 17, 1 to 12, 1 to 6, 1 to 3, 1, or 2.
  • the polynucleotide (b2) for example, the 204th to 212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1) above may be further preserved.
  • the polynucleotide of (b2) is, for example, the 45th base (A), 48th base (A), 49th base (T), 51st base (T) in (b1).
  • a polynucleotide comprising a nucleotide sequence in which a nucleotide sequence (5'-AAAAGCTCCA-3 ') is conserved and one or several bases are deleted, substituted, inserted and / or added in a nucleotide sequence other than the aforementioned nucleotide sequence .
  • the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the polynucleotide (b3) for example, the 204th to 212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1) above may be further preserved.
  • the polynucleotide of (b3) is, for example, 45th base (A), 48th base (A), 49th base (T), 51st base (T) in (b1).
  • a polynucleotide comprising a nucleotide sequence having a nucleotide sequence (5′-AAAAGCTCCA-3 ′) conserved and having 80% or more identity to a nucleotide sequence other than the aforementioned nucleotide sequence.
  • the resistance locus When the resistance locus satisfies at least one of the conditions (5) and (6), the resistance locus may satisfy, for example, one of the conditions (5) and (6). Both conditions may be satisfied, and it is preferable to satisfy both conditions because of higher correlation with powdery mildew resistance.
  • the resistance locus may satisfy, for example, at least one of the following conditions (7) and (8) as a condition based on the base sequence.
  • the following (c2) and (c3) are polynucleotides having functions equivalent to those in the following (c1) with respect to the powdery mildew resistance at the resistance locus.
  • the following (d2) and (d3) are polynucleotides having a function equivalent to that of the following (d1) with respect to the powdery mildew resistance at the resistance locus.
  • the powdery mildew resistance locus on chromosome 6 is specified by the polynucleotide (c) below.
  • C Polynucleotide (c2) consisting of the base sequence of SEQ ID NO: 4 (c1), (c1), (c2), or (c3) below The 50th base (C), 141st of (c1) Of the base (A) and the 266th base (T), and a base sequence other than the base, wherein one or several bases are deleted, substituted, inserted and / or added.
  • Nucleotide (c3) The 50th base (C), 141st base (A), and 266th base (T) of (c1) are stored, and 80% or more of the base sequence other than the base A polynucleotide comprising a nucleotide sequence having the same identity
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (d) below.
  • polynucleotide (d2) comprising the nucleotide sequence of SEQ ID NO: 5 (d1), polynucleotide (d1) below (d1), (d2) or (d3) 99th base (C), 174th of (d1) Base (G), 184th base (T), 199th base (A), and 200th base (T) are conserved, and in the base sequence other than the base, one or several bases are missing.
  • Polynucleotide (d3) comprising the nucleotide sequence deleted, substituted, inserted and / or added (d3) 99th base (C), (174) base (G), 184th base (T), 199 A polynucleotide comprising a base sequence having 80% or more identity to a base sequence other than the base, wherein the base (A) and the base 200 (T) are conserved
  • the first, second, and third bases in the underlined brackets in SEQ ID NO: 4 are bases corresponding to the polymorphisms of the SNP2 50 , the SNP2 141 , and the SNP2 266 It is.
  • the polynucleotide (c1) can be obtained, for example, from a melon plant deposited under the deposit number FERM BP-22291 described later.
  • the “one or several” is, for example, 1 to 100, 1 to 75, 1 to 55, 1 to 50, 1 to 25, 1 to 20, -15, 1-10, 1-5, 1-3, 1 or 2.
  • the polynucleotide (c2) for example, the 132nd base (T) and the 457th base (A) in (c1) may be further preserved.
  • the polynucleotide (c2) is, for example, the 50th base (C), the 132nd base (T), the 141st base (A), and the 266th base (T) in (c1).
  • 457, and the 457th base (A) and is a polynucleotide comprising a base sequence in which one or several bases are deleted, substituted, inserted and / or added in a base sequence other than the base.
  • the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the polynucleotide (c3) for example, the 132nd base (T) and the 457th base (A) in (c1) may be further preserved.
  • the polynucleotide of (c3) is, for example, the 50th base (C), the 132nd base (T), the 141st base (A), and the 266th base (T) in (c1).
  • 457 and the 457th base (A) and is a polynucleotide comprising a base sequence having 80% or more identity to a base sequence other than the base.
  • the first to fourth and fifth bases in the underlined brackets in SEQ ID NO: 5 are the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 It is a base corresponding to the polymorphism of.
  • the polynucleotide (d1) can be obtained, for example, from a melon plant deposited under the deposit number FERM BP-22291 described later.
  • the “one or several” is, for example, 1 to 93, 1 to 70, 1 to 51, 1 to 48, 1 to 24, 1 to 19, -15, 1-10, 1-5, 1-3, 1 or 2.
  • the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
  • the combination of the conditions satisfied by the resistance locus is not particularly limited.
  • the following combinations It can be illustrated.
  • the resistance locus satisfies the condition based on the combination of the length (i) of the amplified fragment and the SNP, but the resistance locus is not limited to this, and as described above, Instead of the condition (i) based on the combination of the length of the amplified fragment and the SNP, the condition (ii) based on the base sequence may be satisfied.
  • the resistance locus may satisfy at least one of the conditions (7) and (8) instead of at least one of the conditions (5) and (6).
  • the resistance locus may satisfy one of the conditions (7) and (8), or may satisfy both conditions, and correlation with powdery mildew resistance. It is preferable that both conditions are satisfied because the property is higher.
  • the resistance locus is, for example, at least one of the base sequence of the amplified fragment amplified with the primer set and the base sequence including the combination of SNPs.
  • the condition based on the base sequence of the region including may be satisfied, and specifically, the following condition (9) may be satisfied.
  • the region may be, for example, a condition including the entire base sequence of the amplified fragment and at least one of the base sequences including the combination of the SNPs or a condition including a part thereof.
  • the powdery mildew resistance locus on the chromosome 6 is identified by the base sequence between two SNP sites selected from the group consisting of the following SNPs (A), (B) and (C) in the chromosome: Is done.
  • A One SNP selected from the group consisting of 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th SNPs in the base sequence of SEQ ID NO: 3
  • B One SNP selected from the group consisting of 50th, 132, 141, 266, and 457th SNPs in the base sequence of SEQ ID NO: 4
  • C One SNP selected from the group consisting of the 99th, 174, 184, 199, and 200th SNPs in the base sequence of SEQ ID NO: 5
  • the 45th , 48th , 49th , 51st , 108th, 120th, 139th, 214th, and 327th SNPs are, for example, the SNP1 45 , the SNP1 48 , the SNP1 49 , and the SNP1 51, respectively.
  • SNP1 108 , SNP1 120 , SNP1 139 , SNP1 214 , and SNP1 327 bases corresponding to polymorphisms.
  • the 50 , 132 , 141 , 266 , and 457th SNPs are, for example, polymorphisms of the SNP2 50 , the SNP2 132 , the SNP2 141 , the SNP2 266 , and the SNP2 457 , respectively. Is a base corresponding to
  • the 99 , 174 , 184 , 199 , and 200th SNPs are, for example, polymorphisms of the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 , respectively. Is a base corresponding to
  • the region identifies an upstream end and a downstream end by two SNP sites selected from the group consisting of SNPs (A), (B), and (C). it can.
  • the region may be, for example, between two SNP sites selected from the group consisting of the SNPs of (A), (B) and (C), for example, both or one of the two SNP sites. May or may not be included.
  • the upstream end and the downstream end of the region are the SNP part, but the upstream end and the downstream end.
  • the base may be, for example, a base surrounded by an underline or a square in the base sequence described above, or may be a base other than that.
  • the SNP between the upstream end and the downstream end can be determined based on the seating positions of the SNPs (A), (B), and (C), for example.
  • the SNPs of (A), (B), and (C) are, for example, from the upstream side (SNP3 99 ) to the downstream side (SNP2 457 ) on chromosome 6 of the melon plant, as shown in FIG.
  • the SNP of (C), the SNP of (A), and the SNP of (B) are seated in this order.
  • the base sequence of the amplified fragment is located between, for example, the SNP of (A) and the SNP of (B).
  • examples of the two SNPs that specify the region include the following combinations.
  • Combination of SNP of (A) and SNP of (B) Combination of SNP of (A) and SNP of (C)
  • Combination of SNP of (B) and SNP of (C) Of the above combinations, powdery mildew Since the correlation with the resistance is higher, for example, the following combinations are preferable.
  • the resistance locus when the resistance locus is specified by the base sequence of the region between the two SNP sites, it is preferable that the resistance locus further includes the above-described conditions related to the base sequence of the region. Specifically, the resistance locus preferably satisfies, for example, at least one of the conditions (1) and (2) in the base sequence of the region. Moreover, it is preferable that the resistance locus further satisfies, for example, at least one of the conditions (3) and (4) in the base sequence of the region.
  • Examples of the related conditions include conditions related to the base sequence between the SNP site at the upstream end and the SNP site at the downstream end that specify the region, and are shown in FIG. It can be determined appropriately based on the SNPs of (A), (B) and (C) and the locus position of the base sequence of the amplified fragment.
  • the number of the related conditions may be one or more, for example, and as a specific example, is all conditions related to the base sequence seated between the SNP sites specifying the region.
  • the combination of the base sequence of the region between the two SNP sites satisfied by the resistance locus and the conditions related to the base sequence of the region is not particularly limited.
  • Condition (a) The powdery mildew resistance locus on the chromosome 6 comprises the nucleotide sequence of the region between the sites of the SNP of (A) and the SNP of (B) in the chromosome, and Satisfy at least one of the conditions (1) and (2).
  • the powdery mildew resistance locus on the chromosome 6 comprises the nucleotide sequence of the region between the site of the SNP of (B) and the SNP of (C) in the chromosome, and Satisfy at least one of the conditions (1) and (2).
  • Condition (c) The powdery mildew resistance locus on the chromosome 6 comprises the nucleotide sequence of the region between the site of the SNP of (B) and the SNP of (C) in the chromosome, and At least one of the conditions (1) and (2) and at least one of the conditions (3) and (4) are satisfied.
  • the resistance locus may satisfy, for example, one of the conditions (1) and (2), or may satisfy both conditions.
  • the resistance locus may satisfy one of the conditions (1) and (2), or may satisfy both conditions.
  • the combination of conditions satisfied by the resistance locus is not particularly limited, and examples thereof include the following combinations.
  • the resistance marker of the present invention for example, powdery mildew resistance can be imparted to melon plants.
  • the degree of powdery mildew resistance of a melon plant can be represented by, for example, a disease index by referring to the method described in Reference Document 2 below.
  • a disease index of 1 or less can be set as disease resistance (resistance), and a disease index of 2 or more can be set as susceptibility.
  • the resistance marker of the present invention may further include another resistance marker, for example.
  • the powdery mildew resistant melon plant of the present invention comprises a powdery mildew resistant gene locus on chromosome 6 in a homozygous form as described above, The powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
  • Condition (1) The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1, The length of the amplified fragment is 133 bases or more.
  • Primer set 1 Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
  • Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  • the powdery mildew resistant melon plant of the present invention comprises the powdery mildew resistant locus on chromosome 6 in a homozygous form, and the powdery mildew resistant locus on chromosome 6 has the following conditions ( It is characterized in that at least one of the conditions 1) and (2) is satisfied, and other configurations and conditions are not particularly limited. Since the powdery mildew resistant melon plant of this invention contains the resistance marker of the said this invention which is the said resistance locus, description of the said resistance marker of this invention can be used, for example. In the present invention, the powdery mildew resistance locus on chromosome 6 can be read as the resistance locus in the resistance marker of the present invention, for example. For the powdery mildew resistant melon plant of the present invention, for example, the description of the resistance marker of the present invention can be incorporated.
  • the powdery mildew resistant melon plant of the present invention is resistant to powdery mildew.
  • the powdery mildew resistance is brought about by the resistance locus.
  • the powdery mildew resistant melon plant of the present invention includes the resistance locus on the sixth chromosome in a homozygous form, but the powdery mildew resistant melon plant includes, for example, a chromosome 6 instead of the sixth chromosome.
  • the resistance locus on the sixth chromosome may be included on any chromosome other than the sixth chromosome.
  • the powdery mildew resistant melon plant has chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 7, chromosome 8, chromosome 9, chromosome 10,
  • the resistance locus on the sixth chromosome may be included on either the chromosome or the twelfth chromosome.
  • the powdery mildew resistant melon plant includes the resistance marker on a chromosome other than the sixth chromosome, for example, one resistance locus may be included on a chromosome other than the sixth chromosome, or two resistances may be included. Sex loci may be included on chromosomes other than chromosome 6.
  • the powdery mildew resistant melon plant may include, for example, the two resistance loci on the same chromosome or different chromosomes.
  • the “chromosome” can also be referred to as a “linkage group” as described above, for example, and the above description can be used.
  • the description of the resistance marker of the present invention can be cited as the resistance locus, for example.
  • the powdery mildew resistant melon plant of the present invention may satisfy, for example, one of the conditions (1) and (2), or may satisfy both conditions, and correlation with powdery mildew resistance. It is preferable that both conditions are satisfied because the property is higher.
  • An example of the powdery mildew resistant melon plant of the present invention is a melon plant ( Cucumis melo ) deposited under the accession number FERM BP-22291 or its progeny line.
  • the deposited melon plant includes, for example, the resistance locus on the sixth chromosome in a homozygous form.
  • the deposit information is shown below.
  • Deposit Type International Depositary Agency Name: National Institute of Technology and Evaluation Patent Biological Depositary Center Address: Japan 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818 Room No. 120 Accession Number: FERM BP- 22291 Display for identification: Takii8 Date of receipt: August 14, 2015
  • the powdery mildew resistant melon plant of the present invention can also be produced, for example, by introducing the resistance locus into a melon plant.
  • the method for introducing the resistance locus into the melon plant is not particularly limited, and examples thereof include conventionally known genetic engineering techniques. Examples of the resistance locus to be introduced include the powdery mildew resistance locus described above.
  • powdery mildew resistant melon plant of the present invention characteristics other than powdery mildew resistance, such as trait and ecological characteristics are not particularly limited.
  • the powdery mildew resistant melon plant of the present invention may further have other resistance.
  • the “plant body” may mean either a plant individual indicating the whole plant or a part of the plant individual.
  • the plant individual part include organs, tissues, cells, vegetative propagation bodies, and the like.
  • the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like.
  • the tissue is, for example, a part of the organ.
  • the plant body part may be, for example, one kind of organ, tissue and / or cell, or two or more kinds of organ, tissue and / or cell.
  • the powdery mildew resistant melon plant of the present invention (hereinafter also referred to as “manufacturing method”) will be described.
  • the following methods are examples, and the present invention is not limited to these methods.
  • the production method can also be referred to as a growth method, for example.
  • the powdery mildew resistance locus can be rephrased as the resistance marker of the present invention.
  • the method for producing a powdery mildew resistant melon plant of the present invention includes the following steps (a) and (b).
  • the production method of the present invention is characterized by using the powdery mildew resistant melon plant of the present invention as a parent, and other steps and conditions are not particularly limited.
  • the description of the resistance marker of the present invention can be used in the production method of the present invention.
  • the powdery mildew resistance locus can impart powdery mildew resistance at a single locus, for example.
  • the production method of the present invention for example, by using the above-mentioned resistance locus, can easily produce progeny showing powdery mildew resistance from F1 obtained by crossing with other melon plants or from its progeny. Can get to.
  • the powdery mildew resistant melon plant used as the first parent may be the powdery mildew resistant melon plant of the present invention.
  • the powdery mildew resistant melon plant is preferably, for example, a melon plant deposited under the accession number FERM BP-22291 as described above or its progeny line.
  • the powdery mildew resistant melon plant used as the first parent can be obtained, for example, by the screening method of the present invention described later. Therefore, the powdery mildew resistant melon plant is obtained, for example, from the test melon plant (hereinafter also referred to as “candidate melon plant”) by the following step (x) prior to the step (a). You may select and prepare.
  • (X) a step of selecting the powdery mildew resistant melon plant of the present invention from the test melon plant
  • step (x) selection of the powdery mildew resistant melon plant can be said to be selection of a melon plant containing the resistant locus.
  • the said (x) process can be performed by the following (x1) process and (x2) process, for example.
  • (X1) a detection step of detecting the presence or absence of a homozygous powdery mildew resistance locus on the chromosome of the test melon plant (x2) due to the presence of the homozygous powdery mildew resistance locus, Selection process for selecting the test melon plant as a powdery mildew resistant melon plant
  • the selection in the step (x) is, for example, selection of a melon plant containing the powdery mildew resistance locus, and specifically, for the test melon plant, the powdery mildew By detecting the resistance locus, the powdery mildew resistant melon plant can be selected.
  • the detection of the powdery mildew resistance locus is, for example, a condition based on a combination of the length of the amplified fragment and the SNP that the resistance locus satisfies, as described in the resistance marker of the present invention. , (Ii) conditions based on the base sequence, (iii) conditions based on the base sequence of the region, and combinations of these conditions.
  • the selection in the step (x) is, for example, selection of a powdery mildew resistant melon plant containing a powdery mildew resistant locus on chromosome 6 in a homozygous form, and powdery mildew on the sixth chromosome.
  • the disease resistance locus satisfies at least one of the conditions (1) and (2).
  • the resistance locus is selected based on, for example, (i) the condition based on at least one of the length of the amplified fragment amplified by the primer set and the combination of SNPs.
  • the resistance locus may be selected, for example, under conditions based on at least one of the base sequence of the amplified fragment amplified by the primer set and the base sequence including the combination of SNPs, as will be described later.
  • (Iii) may be selected based on conditions based on the base sequence of the amplified fragment amplified by the primer set and the base sequence of the region containing at least one of the base sequences including the combination of SNPs, or depending on the conditions of these combinations It may be selected.
  • the resistance locus is selected by, for example, (i).
  • the present invention is not limited to this, and for example, instead of (i), by (ii) or (iii) It may be selected, or may be selected according to a combination of (ii) and (iii). Combination of (i) and (ii) Combination of (i) and (iii) Combination of (i), (ii), and (iii)
  • conditions based on the combination of length of amplified fragment and SNP used for the selection are not particularly limited, For example, the description of “(i) Conditions based on a combination of length of amplified fragment and SNP” in the resistance marker of the present invention can be cited.
  • the selection in the step (x) may be, for example, selection of a powdery mildew resistant melon plant that satisfies one of the conditions (1) and (2), or a powdery mildew that satisfies both conditions. It is also possible to select a disease-resistant melon plant.
  • the resistance locus is further specified by, for example, the 204th to 212th and 213th base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in the base sequence of SEQ ID NO: 3. May be.
  • the resistance locus may further satisfy at least one of the conditions (3) and (4) as a condition based on the combination of the SNPs.
  • the resistance locus may be further specified by, for example, a combination of the 132nd and 457th SNPs in the base sequence of SEQ ID NO: 4.
  • the combination of the conditions satisfied by the resistance locus is not particularly limited. Examples of combinations in resistance markers can be cited.
  • the resistance locus satisfies, for example, at least one of the conditions (1) and (2), but the resistance locus is not limited thereto, and the conditions Instead of at least one of the conditions (1) and (2), at least one of the conditions (3) and (4) may be satisfied.
  • the resistance locus may satisfy one of the conditions (3) and (4), or may satisfy both conditions, and has a correlation with powdery mildew resistance. Since it is higher, it is preferable to satisfy both conditions.
  • step (Ii) Selection based on conditions based on nucleotide sequence Selection in the step (x) is, for example, selection of a powdery mildew resistant melon plant containing the powdery mildew resistant gene locus on chromosome 6 in a homozygous form. Yes, the powdery mildew resistance locus on the sixth chromosome may satisfy at least one of the conditions (5) and (6).
  • conditions based on (ii) base sequence used for the selection are not particularly limited, and for example, the description of “(ii) conditions based on base sequence” in the resistance marker of the present invention is used. it can.
  • the selection in the step (x) may be, for example, selection of a powdery mildew-resistant melon plant that satisfies one of the conditions (5) and (6), or an noodle that satisfies both conditions It is also possible to select a disease-resistant melon plant.
  • the polynucleotide (b2) further stores, for example, the 204th to 212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1). May be.
  • the polynucleotide (b3) further includes, for example, the 204-212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1). It may be saved.
  • the resistance locus may further satisfy at least one of the conditions (7) and (8) as a condition based on the base sequence, for example.
  • the condition (7) for example, in the polynucleotide (c2), the 132nd base (T) and the 457th base (A) in the (c1) may be further preserved.
  • the polynucleotide (c3) may further store, for example, the 132nd base (T) and the 457th base (A) in the (c1).
  • the combination of the conditions satisfied by the resistance locus is not particularly limited. Examples of combinations in resistance markers can be cited.
  • the selection in the step (x) is, for example, that of a powdery mildew resistant melon plant containing the powdery mildew resistant locus on the sixth chromosome in a homozygous form.
  • the powdery mildew resistance locus on the sixth chromosome may be selected, and the condition (9) may be satisfied.
  • the conditions based on the nucleotide sequence of the (iii) region used for the selection are not particularly limited. For example, “conditions based on the nucleotide sequence of the (iii) region” in the resistance marker of the present invention. Can be used.
  • the resistance locus preferably satisfies at least one of the conditions (1) and (2) in the base sequence of the region. Moreover, it is preferable that the resistance locus further satisfies, for example, at least one of the conditions (3) and (4) in the base sequence of the region.
  • the resistance locus may satisfy, for example, the condition (a), (b), or (c).
  • the chromosome for detecting the presence or absence of the homozygous resistance locus is preferably the sixth chromosome.
  • the melon plant used as the other parent is not particularly limited.
  • a melon plant containing a known powdery mildew resistance gene may be used, or a melon plant having other resistance.
  • the powdery mildew resistant melon plant of the present invention may be used.
  • the method of crossing the powdery mildew resistant melon plant with the other melon plant is not particularly limited, and a known method can be adopted.
  • the target for selecting the powdery mildew resistant melon plant may be, for example, a melon plant obtained from the step (a) or a progeny line obtained from the melon plant. .
  • the target may be, for example, an F1 melon plant obtained by crossing in the step (a) or a progeny line.
  • the progeny line may be, for example, an inbred progeny or a backcross progeny of the F1 melon plant obtained by crossing in the step (a), or crossing the F1 melon plant with another melon plant. It may be a melon plant obtained by
  • step (b) selection of powdery mildew resistant melon plants can be performed, for example, by confirming powdery mildew resistance directly or indirectly.
  • the direct confirmation can be performed by, for example, evaluating the powdery mildew resistance of the obtained F1 melon plant or its progeny line by the disease incidence index as described above.
  • powdery mildew fungus is inoculated to the F1 melon plant or its progeny line, and powdery mildew resistance can be confirmed by evaluating the disease index.
  • the F1 melon plant having a disease severity of 1 or less or its progeny can be selected as a powdery mildew resistant melon plant.
  • the selection by the indirect confirmation can be performed by, for example, the following steps (b1) and (b2).
  • B2) Powdery mildew resistance A selection step of selecting the melon plant obtained by the step (a) or its progeny line as a powdery mildew resistant melon plant due to the presence of the sex locus
  • the selection of the powdery mildew resistant melon plant in the step (b) is, for example, the same as the method described in the step (x), by detecting the presence or absence of the homozygous powdery mildew resistant locus. More specifically, the detection can be performed by detecting the presence or absence of the homozygous powdery mildew resistance locus using the molecular marker.
  • the melon plant or its progeny line confirmed to be resistant to powdery mildew can be selected as a powdery mildew resistant melon plant.
  • the production method of the present invention may further include a seeding step of collecting seeds from the progeny line obtained by crossing.
  • Screening Method for Powdery Mildew Resistant Melon Plant is for producing a powdery mildew resistant melon plant by crossing.
  • a parent including a process for selecting a powdery mildew resistant melon plant containing a powdery mildew resistance locus on chromosome 6 in homozygous form as a powdery mildew resistance marker of a melon plant from a test melon plant .
  • the powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
  • the powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1, The length of the amplified fragment is 133 bases or more.
  • Primer set 1 Forward primer consisting of the base sequence of SEQ ID NO: 1
  • Reverse primer condition consisting of the base sequence of SEQ ID NO: 2
  • the powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  • a powdery mildew resistant melon plant containing a powdery mildew resistance locus on chromosome 6 in a homozygous form as a powdery mildew resistance marker of a melon plant is selected from test melon plants.
  • the powdery mildew resistance locus on chromosome 6 satisfies at least one of the conditions (1) and (2), and the other steps and conditions are particularly limited.
  • a powdery mildew resistant parent can be obtained by the resistance marker of the present invention.
  • the description of the resistance marker of the present invention can be used.
  • step (x) in the method for producing a powdery mildew resistant melon plant of the present invention can be used.
  • Example 1 Analysis of the inheritance pattern of the powdery mildew resistance locus, identification of the powdery mildew resistance locus, and the powdery mildew resistance locus and powdery mildew resistance of the novel powdery mildew resistant melon plant
  • the powdery mildew resistance locus is a powdery mildew resistance marker for melon plants
  • the melon plant containing the resistance locus is a powdery mildew resistant melon plant. It was confirmed that the powdery mildew resistant melon plant can be screened using the powdery mildew resistance marker of the melon plant.
  • the deposited lines are the SNP1 45 , SNP1 48 , SNP1 49 , SNP1 51 , SNP1 108 , SNP1 120 , SNP1 139 , SNP1 214 , SNP1 327 , SNP2 50 , SNP2 132 , SNP2 141 , SNP2 266 , and SNP2 457 , and SNP3 99 , SNP3 174 , SNP3 184 , SNP3 199 , and SNP3 200 in a resistive homojunction type . That is, the deposited line is a melon plant that satisfies the conditions (1) to (8) and (9).
  • two chromosomes 6 include the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3, and the length of the amplified fragment amplified by the primer set is as follows: 133 bases.
  • the susceptible melon plant is the SNP1 45 , SNP1 48 , SNP1 49 , SNP1 51 , SNP1 108 , SNP1 120 , SNP1 139 , SNP1 214 , and SNP1 327 on chromosome 6.
  • the susceptible melon plant has two chromosomes 6 that do not contain the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3, and the length of the amplified fragment amplified by the primer set. Is 132 bases. That is, the susceptible melon plant is a melon plant that does not satisfy the conditions (1) to (8) and (9).
  • the powdery mildew strain 1 (Race 3.5) was cultured on a melon plant (Otome no Prayer, Takii Seed Co., Ltd.) grown in an isolated incubator. After collecting the conidia by scraping the flora of the powdery mildew fungus on the leaves of the melon plant, sterile distilled water was added. After the addition, the resulting suspension was filtered through Kimwipe (registered trademark), and the filtrate was recovered. Further, the conidia were diluted with sterilized distilled water so as to be 5 ⁇ 10 4 cells / mL to prepare a conidia suspension, which was used as an inoculation source.
  • the 61 strains were previously planted in rice husk charcoal and then potted in 1 / 10,000 Wagner pots filled with sterilized soil.
  • a melon plant in the third development stage of the main leaf was used.
  • the conidial suspension was sprayed uniformly over the entire strain of melon plants.
  • the melon plant was grown for 2 weeks in an artificial weather room under conditions of 20-25 ° C., humidity 60-80%, 14000 Lux, 12 hours of day length. And about the grown melon plant, the disease investigation was conducted as follows. In addition, the pathogenesis of the 10 deposited lines and the 10 susceptible melon plants were similarly investigated.
  • FIG. 2 shows the evaluation index of the disease index as the disease index 0 (FIG. 2A), disease index 1 (FIG. 2B), disease index 2 (FIG. 2C), and disease index.
  • 3 shows a photograph of a representative example of leaves of a melon plant. In each figure, the area where spore formation is observed is the enclosed area indicated by the arrow.
  • Disease index 0 No sporulation observed (high resistance)
  • Disease index 1 Slight sporulation is observed (resistance)
  • Onset index 2 Onset of limited sporulation
  • Onset index 3 Extensive sporulation over a wide range
  • the powdery mildew resistant melon plant has a length of 133 bases of the amplified fragment amplified from two chromosomes 6 by the primer set 1,
  • the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , the SNP1 120 , the SNP1 139 , the SNP1 214 , and the SNP1 327 are respectively A, A, T, T, T, A, A, T, and C
  • the two chromosomes 6 include the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3, respectively. I understood it.
  • the 61 lines of DNA were subjected to PCR using the primer set 1, and the length of the amplified fragment was measured. Further, PCR was performed on the 61 strains of DNA using the primer set 2 below, and polymorphic bases corresponding to the first SNP set were identified in the amplified fragments.
  • the amplification using the primer set 1 was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 57 ° C. for 30 seconds, and 72 ° C. for 60 seconds. This was carried out by incubating at 3 ° C. for 3 minutes.
  • the amplification using the primer set 2 was carried out at 94 ° C.
  • the 61 strains are resistant homozygous (A), heterozygous (H), and susceptible homozygous. Classified into (B). These results are shown in Table 2 below.
  • A represents an individual in which the lengths of the amplified fragments amplified from two chromosomes 6 are 133 bases or more and the first SNP set is a resistant homozygous type. Show.
  • the length of the amplified fragment amplified from one chromosome 6 is 133 bases or more, and the length of the amplified fragment amplified from the other chromosome 6 is less than 133 bases, and H 3 shows an individual having the first SNP set in a heterozygous form.
  • B shows the individual
  • Primer set 2 Forward primer 2 5'-GGAAAAATGCAGGGGAAG-3 '(SEQ ID NO: 8) Reverse primer 2 5'-CTGCCAAAAGCGACTTAACC-3 '(SEQ ID NO: 9)
  • the resistance locus specified by the length of the amplified fragment and the first SNP set are responsible for powdery mildew resistance.
  • the resistance locus specified by the length of the amplified fragment and the first SNP set that is, the resistance locus satisfying both of the conditions (1) and (2) has resistance to powdery mildew. Therefore, the resistance locus serves as a powdery mildew resistance marker for melon plants, and powdery mildew resistant melon plants are screened using the powdery mildew resistance marker for melon plants. I knew it was possible.
  • n 0 , n 1 , n 2 , n 3 indicates the number of individuals of the disease index 0, the disease index 1, the disease index 2, and the disease index 3, respectively.
  • Vedrantais and PMR45 which are known powdery mildew susceptible melon plants, and the susceptible melon plant showed susceptibility to any powdery mildew fungus.
  • the known powdery mildew resistant melon plants PMR5 and PI124112 showed resistance to the powdery mildew strains 2 and 3, whereas the powdery mildew strains 1 and 4 were resistant to the powdery mildew strain 1 and 4. Showed susceptibility.
  • WMR29 and Edisto47 were resistant to the powdery mildew strain 2 whereas the powdery mildew strains 1, 3, and 4 were susceptible.
  • PI1414723 was resistant to the powdery mildew strain 1 to 3, whereas it was susceptible to the powdery mildew strain 4.
  • the MR1 showed resistance to the powdery mildew strains 2 to 4, whereas it showed susceptibility to the powdery mildew strain 1.
  • the F3 selection line and the deposited line showed resistance to any powdery mildew.
  • the powdery mildew strains 1 to 4 are all powdery mildews capable of infecting melon plants containing a known powdery mildew resistance gene
  • the resistance locus is a known powdery mildew. It was also found effective against powdery mildew fungus capable of infecting melon plants containing the disease resistance gene. From these results, it was found that the F3 selection line and the deposited line are resistant to a plurality of known powdery mildew fungi of races.
  • known powdery mildew resistant melon plants show susceptibility to any of powdery mildew strains 1 to 4, whereas the F3 selected strain and the deposited strain are powdery mildew strains 1 to 4. Resistance to any of 4 was shown. From these results, a novel powdery mildew resistance gene in which the resistance locus satisfying both conditions (1) and (2) is different from the resistance locus contained in the known powdery mildew resistant melon plant It was confirmed that it was a seat.
  • Powdery mildew resistance locus on chromosome 6 F2 segregated population was prepared in the same manner as (2) above.
  • the length of the amplified fragment and the polymorphic base corresponding to the first SNP set were identified in the same manner as in (4).
  • PCR was performed on the DNA of the F2 isolated population using the following primer sets 3 and 4, and polymorphic bases corresponding to the second SNP set and the third SNP set were identified in the amplified fragments.
  • the amplification using the primer set 3 was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 51 ° C. for 30 seconds, and 72 ° C. for 60 seconds.
  • Primer set 3 Forward primer 3 5'-AGGAAACGAAGAATAGACG-3 '(SEQ ID NO: 10) Reverse primer 3 5'-TGAGAACCGGAAAGAGAAGC-3 '(SEQ ID NO: 11)
  • Primer set 4 Forward primer 4 5'-GTTCGGATCGGAAAATTCAA-3 '(SEQ ID NO: 12)
  • Reverse primer 4 5'-CCAAGCTTTCCGACATTCAT-3 '(SEQ ID NO: 13)
  • genotypes were classified for the length of the amplified fragment of the F2 segregated population, the first SNP set, the second SNP set, and the third SNP set. Moreover, about the said F2 isolation
  • A indicates that the lengths of the amplified fragments amplified from the two chromosomes 6 by the primer set 1 are both 133 bases or more
  • H is: The length of the amplified fragment amplified from one chromosome 6 with the primer set 1 is 133 bases or more, and the length of the amplified fragment amplified from the other chromosome with the primer set 1 is less than 133 bases
  • B indicates that the lengths of the amplified fragments amplified from two chromosomes 6 are both less than 133 bases.
  • A indicates that each SNP set is a resistant homozygous type
  • H indicates that each SNP set is a heterozygous type.
  • H indicating that each SNP set is susceptible to homozygosity.
  • A is shaded.
  • the disease index of each individual was 1 or less. From these results, the length of the amplified fragment and the first SNP set show high correlation with the powdery mildew resistance at the resistance locus, that is, the conditions (1) and (2) The condition was highly correlated with the powdery mildew resistance. In addition, since it shows a high correlation with the length of the amplified fragment and the first SNP set, the region between the SNP sites of the second SNP set and the third SNP set, which are regions including these, It was found that there was a high correlation with powdery mildew resistance, that is, the condition (9) was a condition highly correlated with the powdery mildew resistance.
  • the resistance locus satisfying at least one of the conditions (1) and (2) and the resistance locus satisfying the condition (9) become a powdery mildew resistance marker of melon plants. It was also found that the powdery mildew resistant melon plant can be screened using the powdery mildew resistance marker of the melon plant.
  • powdery mildew resistant melon plants of the present invention for example, powdery mildew resistant melon plants can be easily screened. Moreover, since the powdery mildew resistant melon plant of this invention contains the said powdery mildew resistant gene locus, for example, it can show powdery mildew resistant, for example. Furthermore, the powdery mildew resistance locus can confer powdery mildew resistance at, for example, a single locus. For this reason, the powdery mildew resistant melon plant of this invention can obtain easily the progeny which shows powdery mildew resistance from F1 obtained by crossing with another melon plant, or its progeny, for example. .
  • the melon plant containing the powdery mildew resistance marker also exhibits resistance to powdery mildew bacteria that can infect melon plants containing the powdery mildew resistance gene of the prior art document, for example.
  • the powdery mildew resistant melon plant of this invention does not need the control by the conventional agrochemical, the problem of the labor and cost of the said agrochemical spraying can also be avoided, for example.

Abstract

Provided are: a novel white mildew resistance marker for melon plants; a white mildew-resistant melon plant carrying a white mildew resistance gene locus; and a method for producing a white mildew-resistant melon plant using the aforementioned white mildew-resistant melon plant. The white mildew resistance marker for melon plants according to the present invention contains a white mildew resistance gene locus on chromosome-6 in a homozygous state, said marker being characterized in that the white mildew resistance gene locus on chromosome-6 meets at least one of the following requirements (1) and (2). (1) The white mildew resistance gene locus on chromosome-6 is specified by the length of an amplification fragment that is amplified using the below-mentioned primer set 1, wherein the length of the amplification fragment is 133 nucleotides or longer: the primer set 1 is composed of a forward primer 1 comprising the nucleotide sequence represented by SEQ ID NO: 1 and a reverse primer 1 comprising the nucleotide sequence represented by SEQ ID NO: 2. (2) The white mildew resistance gene locus on chromosome-6 is specified by polymorphisms in nucleotides located at position-45, position-48, position-49, position-51, position-108, position-120, position-139, position-214 and position-327 in the nucleotide sequence represented by SEQ ID NO: 3.

Description

メロン植物のうどんこ病抵抗性マーカー、うどんこ病抵抗性メロン植物、およびそれを用いたうどんこ病抵抗性メロン植物の製造方法Powdery mildew resistance marker of melon plant, powdery mildew resistant melon plant, and method for producing powdery mildew resistant melon plant using the same
　本発明は、メロン植物のうどんこ病抵抗性マーカー、うどんこ病抵抗性メロン植物、およびそれを用いたうどんこ病抵抗性メロン植物の製造方法に関する。 The present invention relates to a powdery mildew resistance marker for melon plants, a powdery mildew resistant melon plant, and a method for producing a powdery mildew resistant melon plant using the same.
　メロン栽培において、うどんこ病菌による病害は、世界的に深刻な問題となっている。うどんこ病菌に感染した植物体は、葉の枯死等により、植物体の生育が減退し、その結果、果実の減収が生じる。 In melon cultivation, diseases caused by powdery mildew are a serious problem worldwide. The plant body infected with powdery mildew fungus has a reduced growth of the plant body due to leaf death or the like, resulting in a decrease in fruit yield.
　このため、うどんこ病に対する抵抗性遺伝子を利用して、うどんこ病菌に抵抗性を示す品種の育成が試みられている。しかしながら、これらの抵抗性遺伝子を含むメロン植物に対して感染可能なうどんこ病菌が出現し、問題となっている（非特許文献１）。 For this reason, attempts have been made to breed varieties that are resistant to powdery mildew using a powdery mildew resistance gene. However, powdery mildew fungus capable of infecting melon plants containing these resistance genes has emerged and has become a problem (Non-Patent Document 1).
　そこで、本発明は、新たなメロン植物のうどんこ病抵抗性マーカー、うどんこ病抵抗性遺伝子座を含むうどんこ病抵抗性メロン植物、およびそれを用いたうどんこ病抵抗性メロン植物の製造方法の提供を目的とする。 Accordingly, the present invention provides a powdery mildew resistance marker for a new melon plant, a powdery mildew resistant melon plant containing a powdery mildew resistance locus, and a method for producing a powdery mildew resistant melon plant using the same. The purpose is to provide.
　前記目的を達成するために、本発明のメロン植物のうどんこ病抵抗性マーカーは、第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする。
条件（１）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
前記増幅断片の長さが、１３３塩基長以上である。
プライマーセット１
　　　配列番号１の塩基配列からなるフォワードプライマー１
　　　配列番号２の塩基配列からなるリバースプライマー１
条件（２）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。 To achieve the above object, the powdery mildew resistance marker of the melon plant of the present invention comprises a powdery mildew resistance locus on chromosome 6 in a homozygous form,
The powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
Condition (1)
The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
The length of the amplified fragment is 133 bases or more.
Primer set 1
Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
Condition (2)
The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
　本発明のうどんこ病抵抗性メロン植物は、第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする。
条件（１）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
前記増幅断片の長さが、１３３塩基長以上である。
プライマーセット１
　　　配列番号１の塩基配列からなるフォワードプライマー１
　　　配列番号２の塩基配列からなるリバースプライマー１
条件（２）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。 The powdery mildew resistant melon plant of the present invention comprises a powdery mildew resistant locus on chromosome 6 in a homozygous form,
The powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
Condition (1)
The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
The length of the amplified fragment is 133 bases or more.
Primer set 1
Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
Condition (2)
The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
　本発明のうどんこ病抵抗性メロン植物の製造方法は、下記（ａ）および（ｂ）工程を含むことを特徴とする。
（ａ）前記本発明のうどんこ病抵抗性メロン植物と、他のメロン植物とを交雑する工程
（ｂ）前記（ａ）工程より得られたメロン植物またはその後代系統から、うどんこ病抵抗性メロン植物を選抜する工程 The method for producing a powdery mildew resistant melon plant of the present invention includes the following steps (a) and (b).
(A) Step of crossing the powdery mildew resistant melon plant of the present invention with another melon plant (b) Powdery mildew resistance from the melon plant obtained from the step (a) or its progeny line Process for selecting melon plants
　本発明者らは、鋭意研究の結果、メロン植物について、うどんこ病抵抗性を示すうどんこ病抵抗性マーカーとして、新規のうどんこ病抵抗性遺伝子座を見出した。また、前記うどんこ病抵抗性マーカーを含むメロン植物は、うどんこ病抵抗性を示す。このため、本発明のメロン植物のうどんこ病抵抗性マーカーによれば、例えば、うどんこ病抵抗性メロン植物を簡便にスクリーニングできる。また、本発明のうどんこ病抵抗性メロン植物は、例えば、前記うどんこ病抵抗性遺伝子座を含むため、例えば、うどんこ病抵抗性を示すことが可能である。さらに、前記うどんこ病抵抗性遺伝子座は、例えば、単一の遺伝子座（単因子）でうどんこ病抵抗性を付与できる。このため、本発明のうどんこ病抵抗性メロン植物は、例えば、他のメロン植物との交雑によって得られたＦ１またはその後代からも、うどんこ病抵抗性を示す後代を簡便に得ることができる。さらに、前記うどんこ病抵抗性マーカーを含むメロン植物は、例えば、前記先行技術文献のうどんこ病抵抗性遺伝子を含むメロン植物に対して感染可能なうどんこ病菌に対しても抵抗性を示す。このため、本発明のうどんこ病抵抗性メロン植物は、従来のような農薬による防除が不要であるため、例えば、前記農薬散布の労力および費用の問題も回避できる。 As a result of intensive studies, the present inventors have found a novel powdery mildew resistance locus as a powdery mildew resistance marker showing powdery mildew resistance in melon plants. Moreover, the melon plant containing the said powdery mildew resistance marker shows powdery mildew resistance. Therefore, according to the powdery mildew resistance marker of the melon plant of the present invention, for example, powdery mildew resistant melon plants can be easily screened. Moreover, since the powdery mildew resistant melon plant of this invention contains the said powdery mildew resistant gene locus, for example, it can show powdery mildew resistant, for example. Furthermore, the powdery mildew resistance locus can impart powdery mildew resistance with a single locus (single factor), for example. For this reason, the powdery mildew resistant melon plant of this invention can obtain easily the progeny which shows powdery mildew resistance from F1 obtained by crossing with another melon plant, or its progeny, for example. . Furthermore, the melon plant containing the powdery mildew resistance marker also exhibits resistance to powdery mildew bacteria that can infect melon plants containing the powdery mildew resistance gene of the prior art document, for example. For this reason, since the powdery mildew resistant melon plant of this invention does not need the control by the conventional agrochemical, the problem of the labor and cost of the said agrochemical spraying can also be avoided, for example.
図１は、第６染色体におけるＳＮＰ（single　nucleotide　polymorphism）等の相対的な座乗位置を示す模式図である。FIG. 1 is a schematic diagram showing relative seating positions of SNP (single nucleotide polymorphism) and the like on the sixth chromosome. 図２は、実施例１におけるメロン植物の発病指数の評価基準を示す写真である。FIG. 2 is a photograph showing evaluation criteria for the disease index of melon plants in Example 1.
１．メロン植物のうどんこ病抵抗性マーカー
本発明のメロン植物のうどんこ病抵抗性マーカー（以下、「抵抗性マーカー」ともいう。）は、前述のように、第６染色体上のうどんこ病抵抗性遺伝子座（以下、「抵抗性遺伝子座」ともいう。）をホモ接合型で含み、前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする。 1. Powdery mildew resistance marker of melon plant The powdery mildew resistance marker of the melon plant of the present invention (hereinafter also referred to as “resistance marker”) is powdery mildew resistance on chromosome 6 as described above. A locus (hereinafter also referred to as “resistance locus”) in a homozygous form, wherein the powdery mildew resistance locus on chromosome 6 is at least one of the following conditions (1) and (2): It satisfies the conditions.
条件（１）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
前記増幅断片の長さが、１３３塩基長以上である。
プライマーセット１
　　　配列番号１の塩基配列からなるフォワードプライマー１
　　　配列番号２の塩基配列からなるリバースプライマー１ Condition (1)
The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
The length of the amplified fragment is 133 bases or more.
Primer set 1
Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
条件（２）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。 Condition (2)
The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
　本発明のうどんこ病抵抗性マーカーは、第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とし、その他の構成および条件は、特に制限されない。 The powdery mildew resistance marker of the present invention comprises a powdery mildew resistance locus on chromosome 6 in a homozygous form, and the powdery mildew resistance locus on chromosome 6 is the condition (1). And (2) satisfying at least one of the conditions, and other configurations and conditions are not particularly limited.
　本発明において、「メロン植物」は、キュウリ属CucumisのCucumis　meloに分類される植物である。 In the present invention, the “melon plant” is a plant classified as Cucumis melo of the genus Cucumis .
　本発明において、うどんこ病の病原菌は、例えば、Sphaerotheca　fuliginea（「Podosphaera　xanthii」ともいう）、Erysiphe　polygoni等があげられる。 In the present invention, examples of the powdery mildew pathogen include Sphaerotheca fuliginea (also referred to as “ Podosphaera xanthii ”), Erysiphe polygoni, and the like.
　本発明において、「うどんこ病抵抗性」は、例えば、「うどんこ病耐性」ともいう。前記抵抗性は、例えば、うどんこ病の病原菌の感染による病害の発生および進行に対する阻害能または抑制能を意味し、具体的に、例えば、病害の未発生、発生した病害の進行の停止、および、発生した病害の進行の抑制（「阻害」ともいう。）等のいずれでもよい。 In the present invention, “powder resistance” is also referred to as “powder resistance”, for example. The resistance means, for example, the ability to inhibit or suppress the occurrence and progression of disease caused by infection with the powdery mildew pathogen, specifically, for example, the occurrence of no disease, stop of progression of the disease that has occurred, and Any of suppression of the progression of the disease that has occurred (also referred to as “inhibition”) may be used.
　本発明において、「染色体」は、例えば、「連鎖群」ということもできる。このため、本発明において、前記メロン植物の第１～１２染色体は、それぞれ、例えば、第１～１２連鎖群ということもでき、第６染色体は、例えば、第６連鎖群ということもできる。前記連鎖群は、例えば、メロン植物のゲノムの塩基配列情報に基づき、下記参考文献１を参照し、決定できる。前記メロン植物のゲノムの塩基配列情報は、例えば、メロノミクスプロジェクト（MELONOMICS　Project）のwebサイト（https://melonomics.net/）から入手可能である。具体的に、本発明における前記第６染色体（第６連鎖群）は、例えば、メロノミクスプロジェクトのwebサイトに公開されているメロン植物のゲノムの塩基配列情報Melon_genome_v3.5.1に基づき、下記参考文献１を参照し、決定できる。
参考文献１：Jason M Argyris et.al., “Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly”, BMC Genomics, 2015 vol.16:4 In the present invention, the “chromosome” can also be referred to as a “linkage group”, for example. Therefore, in the present invention, the first to twelfth chromosomes of the melon plant can also be referred to as, for example, the first to twelfth linkage groups, and the sixth chromosome can also be referred to as, for example, the sixth linkage group. The said linkage group can be determined with reference to the following reference 1 based on the base sequence information of the genome of a melon plant, for example. The base sequence information of the melon plant genome can be obtained from, for example, the MELONOMICS Project website (https://melonomics.net/). Specifically, the 6th chromosome (sixth linkage group) in the present invention is based on, for example, the base sequence information Melon_genome_v3.5.1 of the melon plant published on the website of the Melonomics Project. Can be determined.
Reference 1: Jason M Argyris et.al., “Use of targeted SNP selection for an improved anchoring of the melon ( Cucumis melo L.) scaffold genome assembly”, BMC Genomics, 2015 vol.16: 4
　本発明の抵抗性マーカーは、前述のように、前記第６染色体上の抵抗性遺伝子座をホモ接合型で含むが、前記うどんこ病抵抗性メロン植物は、例えば、第６染色体に代えて、第６染色体以外のいずれの染色体上に、前記第６染色体上の抵抗性遺伝子座を含んでもよい。すなわち、前記うどんこ病抵抗性メロン植物は、第１染色体、第２染色体、第３染色体、第４染色体、第５染色体、第７染色体、第８染色体、第９染色体、第１０染色体、第１１染色体、第１２染色体のいずれかの染色体上に、前記第６染色体上の前記抵抗性遺伝子座を含んでもよい。前記うどんこ病抵抗性メロン植物が前記抵抗性マーカーを第６染色体以外の染色体上に含む場合、例えば、１つの抵抗性遺伝子座を第６染色体以外の染色体上に含んでもよいし、２つの抵抗性遺伝子座を第６染色体以外の染色体上に含んでもよい。後者の場合、前記うどんこ病抵抗性メロン植物は、例えば、前記２つの抵抗性遺伝子座を同じ染色体上に含んでもよいし、異なる染色体上に含んでもよい。 As described above, the resistance marker of the present invention contains the resistance locus on the sixth chromosome in a homozygous form, but the powdery mildew resistant melon plant is, for example, replaced with the sixth chromosome, The resistance locus on the sixth chromosome may be included on any chromosome other than the sixth chromosome. That is, the powdery mildew resistant melon plant has chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 7, chromosome 8, chromosome 9, chromosome 10, The resistance locus on the sixth chromosome may be included on either the chromosome or the twelfth chromosome. When the powdery mildew resistant melon plant includes the resistance marker on a chromosome other than the sixth chromosome, for example, one resistance locus may be included on a chromosome other than the sixth chromosome, or two resistances may be included. Sex loci may be included on chromosomes other than chromosome 6. In the latter case, the powdery mildew resistant melon plant may include, for example, the two resistance loci on the same chromosome or different chromosomes.
　うどんこ病抵抗性遺伝子座とは、うどんこ病抵抗性を供与する量的形質遺伝子座または遺伝子領域を意味する。前記量的形質遺伝子座（Quantitative　Traits　Loci　;　QTL）は、一般に、量的形質の発現に関与する染色体領域を意味する。ＱＴＬは、染色体上の特定の座を示す分子マーカーを使用して規定できる。前記分子マーカーを使用してＱＴＬを規定する技術は、当該技術分野において周知である。 The powdery mildew resistance locus means a quantitative trait locus or gene region that provides powdery mildew resistance. The quantitative trait locus (Quantitative Traits Loci; QTL) generally means a chromosomal region involved in the expression of quantitative traits. QTL can be defined using molecular markers that indicate specific loci on the chromosome. Techniques for defining QTL using the molecular markers are well known in the art.
　本発明において、前記うどんこ病抵抗性遺伝子座の規定（以下、「特定」ともいう。）に使用する分子マーカーは、特に制限されない。前記分子マーカーは、例えば、ＳＮＰマーカー、ＡＦＬＰ（分子増幅断片長多型、amplified　fragment　length　polymorphism）マーカー、ＲＦＬＰ（restriction　fragment　length　polymorphism）マーカー、マイクロサテライトマーカー、ＳＣＡＲ（sequence-characterized　amplified　region）マーカーおよびＣＡＰＳ（cleaved　amplified　polymorphic　sequence）マーカー等があげられる。前記マイクロサテライトマーカーは、例えば、ＳＴＲ（short　tandem　repeat）マーカー、ＳＳＲ（simple　sequence　repeat）マーカー等があげられる。前記抵抗性遺伝子座は、例えば、１種類のマーカーで特定されてもよいし、２種類以上のマーカーで特定されてもよい。 In the present invention, the molecular marker used for the regulation (hereinafter also referred to as “specific”) of the powdery mildew resistance locus is not particularly limited. Examples of the molecular markers include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP (restriction fragment length polymorphism) markers, microsatellite markers, SCAR (sequence-characterized amplified region) markers, and CAPS. (Cleaved amplified polymorphic sequence) markers and the like. Examples of the microsatellite marker include a STR (short tandem repeat) marker, a SSR (simple sequence repeat) marker, and the like. The resistance gene locus may be specified by, for example, one type of marker, or may be specified by two or more types of markers.
　本発明において、前記ＳＮＰマーカーは、例えば、１個のＳＮＰを前記ＳＮＰマーカーとしてもよいし、２個以上のＳＮＰの組合せを前記ＳＮＰマーカーとしてもよい。 In the present invention, for the SNP marker, for example, one SNP may be used as the SNP marker, or a combination of two or more SNPs may be used as the SNP marker.
　本発明において、前記抵抗性遺伝子座は、（ｉ）プライマーセットで増幅される増幅断片の長さおよび塩基の多型（以下、「ＳＮＰ」ともいう。）の組合せの少なくとも一方に基づく条件を満たす。すなわち、前記抵抗性遺伝子座は、前述のように、前記条件（１）および前記条件（２）の少なくとも一方の条件を満たす。前記抵抗性遺伝子座は、例えば、さらに、後述するように、（ii）プライマーセットにより増幅される増幅断片の塩基配列およびＳＮＰの組合せを含む塩基配列の少なくとも一方に基づく条件を満たしてもよいし、（iii）プライマーセットで増幅される増幅断片の塩基配列およびＳＮＰの組合せを含む塩基配列の少なくとも一方を含む領域の塩基配列に基づく条件を満たしてもよいし、これらの組合せの条件を満たしてもよい。前記組合せの条件を満たす場合、前記組合せは、特に制限されず、例えば、以下の組合せが例示できる。また、前記抵抗性遺伝子座は、前記（ｉ）を満たしているが、本発明はこれに限定されず、例えば、前記（ｉ）に代えて、前記（ii）または前記（iii）を満たしてもよいし、前記（ii）および前記（iii）を組合せた条件を満たしてもよい。
前記（ｉ）および前記（ii）の組合せ
前記（ｉ）および前記（iii）の組合せ
前記（ｉ）、前記（ii）、および前記（iii）の組合せ In the present invention, the resistance locus satisfies the condition based on at least one of the combination of (i) the length of the amplified fragment amplified by the primer set and the base polymorphism (hereinafter also referred to as “SNP”). . That is, as described above, the resistance locus satisfies at least one of the conditions (1) and (2). The resistance locus may satisfy, for example, (ii) a condition based on at least one of a base sequence of an amplified fragment amplified by a primer set and a base sequence including a combination of SNPs, as described later. (Iii) The conditions based on the base sequence of the amplified fragment amplified with the primer set and the base sequence of the region containing at least one of the base sequences including the combination of SNP may be satisfied, or the conditions of these combinations must be satisfied Also good. When the conditions for the combination are satisfied, the combination is not particularly limited, and examples thereof include the following combinations. Further, the resistance locus satisfies the above (i), but the present invention is not limited to this. For example, instead of the above (i), the above (ii) or (iii) is satisfied. Alternatively, the condition combining (ii) and (iii) may be satisfied.
Combination of (i) and (ii) Combination of (i) and (iii) Combination of (i), (ii), and (iii)
（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件
　前記抵抗性遺伝子座は、前記（ｉ）に示すように、プライマーセットで増幅される増幅断片の長さおよびＳＮＰの組合せの少なくとも一方に基づく条件を満たし、具体的には、前記条件（１）および（２）の少なくとも一方の条件を満たす。以下、各条件における各ＳＮＰは、本発明者らが新たに同定したＳＮＰであり、当該技術分野における当業者であれば、後述するこれらのＳＮＰを含む塩基配列に基づき、前記ＳＮＰの座乗位置を特定できる。 (I) Conditions based on the combination of length of amplified fragment and SNP As shown in the above (i), the resistance locus is included in at least one of the combination of length of amplified fragment and SNP amplified by the primer set. The condition based on this is satisfied, specifically, at least one of the conditions (1) and (2) is satisfied. Hereinafter, each SNP in each condition is a SNP newly identified by the present inventors, and if it is a person skilled in the art, the SNP sitting position based on a base sequence containing these SNPs described later. Can be identified.
　前記条件（１）において、前記抵抗性遺伝子座は、下記プライマーセット１で増幅される増幅断片の長さにより特定される。前記増幅断片の長さは、下記プライマーセット１を用いて、例えば、メロン植物について遺伝子増幅を行い、得られた増幅断片を解析することにより測定できる。前記増幅断片の解析は、例えば、前記増幅断片のシーケンシングでもよいし、電気泳動等による解析でもよい。下記プライマーセット１は、例えば、前述のメロン植物のゲノムの塩基配列情報において、第６染色体上の５８５７０２８～５８５７１５９番目の塩基配列を増幅する。 In the condition (1), the resistance locus is specified by the length of the amplified fragment amplified by the following primer set 1. The length of the amplified fragment can be measured, for example, by performing gene amplification on a melon plant using the following primer set 1 and analyzing the obtained amplified fragment. The analysis of the amplified fragment may be, for example, sequencing of the amplified fragment or analysis by electrophoresis or the like. Primer set 1 below amplifies, for example, nucleotide sequences 5857028 to 5857159 on chromosome 6 in the above-mentioned genome sequence information of the melon plant.
プライマーセット１
　　フォワードプライマー１　5’-AATCTCAACAAGTGAGCTTTTATTGT-3’　　（配列番号１）
　　リバースプライマー１　　5’-CATGATTATCTTCAATTTTCTTTTTGTC-3’　（配列番号２） Primer set 1
Forward primer 1 5'-AATCTCAACAAGTGAGCTTTTATTGT-3 '(SEQ ID NO: 1)
Reverse primer 1 5'-CATGATTATCTTCAATTTTCTTTTTGTC-3 '(SEQ ID NO: 2)
　前記増幅断片の長さは、１３３塩基長以上であればよく、その上限は、特に制限されない。前記増幅断片の長さの上限は、例えば、１４０塩基以下、１３５塩基以下であり、その範囲は、例えば、１３３～１４０塩基長、１３３～１３５塩基長である。前記増幅断片の長さは、好ましくは、１３３塩基長である。なお、前記プライマーセット１で増幅される増幅断片の長さとうどんこ病抵抗性との関連性は、これまでに報告されておらず、本発明者らにより初めて見出された、うどんこ病抵抗性に関与する新規の増幅断片の長さである。 The length of the amplified fragment may be 133 bases or more, and the upper limit is not particularly limited. The upper limit of the length of the amplified fragment is, for example, 140 bases or less and 135 bases or less, and the range thereof is, for example, 133 to 140 bases length, 133 to 135 bases length. The length of the amplified fragment is preferably 133 bases long. The relationship between the length of the amplified fragment amplified with the primer set 1 and the powdery mildew resistance has not been reported so far, and the powdery mildew resistance first discovered by the present inventors. Is the length of the new amplified fragment involved in
　前記条件（２）において、前記抵抗性遺伝子座は、下記配列番号３の塩基配列における９種類のＳＮＰの組合せ（以下、「第１ＳＮＰセット」ともいう。）により特定される。前記９種類のＳＮＰは、下記配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目のＳＮＰである。以下、４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目のＳＮＰを、それぞれ、「ＳＮＰ１４５」、「ＳＮＰ１４８」、「ＳＮＰ１４９」、「ＳＮＰ１５１」、「ＳＮＰ１１０８」、「ＳＮＰ１１２０」、「ＳＮＰ１１３９」、「ＳＮＰ１２１４」、および「ＳＮＰ１３２７」ともいう。前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７は、それぞれ、下記配列番号３の塩基配列におけるかっこで囲んだ下線部の１～８および９番目の塩基である。下記配列番号３の塩基配列は、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。下記配列番号３の塩基配列は、例えば、前述のメロン植物のゲノムの塩基配列情報において、第６染色体上の５５８６９７５～５５８７５４１番目の塩基配列に対応する塩基配列である。 In the condition (2), the resistance locus is specified by a combination of nine types of SNPs in the nucleotide sequence of SEQ ID NO: 3 (hereinafter also referred to as “first SNP set”). The nine types of SNPs are 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th SNPs in the base sequence of SEQ ID NO: 3 below. Hereinafter, the 45th , 48th , 49th , 51st , 108th , 120th, 139th, 214th, and 327th SNPs are respectively referred to as “SNP1 45 ”, “SNP1 48 ”, “SNP1 49 ”, “SNP1 51 ”, “SNP1 108 ”. ”,“ SNP1 120 ”,“ SNP1 139 ”,“ SNP1 214 ”, and“ SNP1 327 ”. The SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , the SNP1 120 , the SNP1 139 , the SNP1 214 , and the SNP1 327 are parentheses in the base sequence of the following SEQ ID NO: 3, respectively. The underlined 1-8 and 9th bases in the box. The base sequence of SEQ ID NO: 3 below can be obtained from, for example, a melon plant deposited under the accession number FERM BP-22291 described later. The base sequence of SEQ ID NO: 3 below is, for example, the base sequence corresponding to the 5586975th to 587541st base sequence on chromosome 6 in the above-mentioned base sequence information of the melon plant genome.
Figure JPOXMLDOC01-appb-I000001
Figure JPOXMLDOC01-appb-I000001
　前記第１ＳＮＰセットは、例えば、前記配列番号３のかっこで囲んだ下線部の塩基において、１～８および９番目の塩基が、それぞれ、Ａ、Ａ、Ｔ、Ｔ、Ｔ、Ａ、Ａ、ＴおよびＣである多型を示す。つまり、例えば、前記第１ＳＮＰセットにおける前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７が、それぞれ、Ａ、Ａ、Ｔ、Ｔ、Ｔ、Ａ、Ａ、Ｔ、およびＣの場合、メロン植物は、うどんこ病抵抗性であり、前記塩基の組合せ以外の塩基の組合せの場合（例えば、Ａ、Ｔ、Ｔ、Ｔ、Ｔ、Ａ、Ｇ、およびＴの場合、Ｇ、Ａ、Ａ、Ａ、Ｇ、Ｃ、Ａ、Ｃ、およびＴの場合）、メロン植物は、うどんこ病罹病性であることを示す。なお、前記第１ＳＮＰセットとうどんこ病抵抗性との関連性は、これまでに報告されておらず、本発明者らにより初めて見出された、うどんこ病抵抗性に関与する新規のＳＮＰの組合せである。 In the first SNP set, for example, in the underlined bases surrounded by the brackets of SEQ ID NO: 3, the 1st to 8th and 9th bases are respectively A, A, T, T, T, A, A, T And polymorphisms that are C. That is, for example, the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , the SNP1 120 , the SNP1 139 , the SNP1 214 , and the SNP1 327 in the first SNP set are respectively A , A, T, T, T, A, A, T, and C, the melon plant is resistant to powdery mildew and in the case of a combination of bases other than the above-mentioned combinations of bases (for example, A, T, In the case of T, T, T, A, G, and T, in the case of G, A, A, A, G, C, A, C, and T), the melon plant is susceptible to powdery mildew. Show. The relationship between the first SNP set and powdery mildew resistance has not been reported so far, and a novel SNP related to powdery mildew resistance, which was first discovered by the present inventors, was found. It is a combination.
　前記条件（２）において、前記抵抗性遺伝子座は、例えば、さらに、前記配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列（配列番号７：5’-AAAAGCTCCA-3’）により特定されてもよい。前記配列番号７の塩基配列は、前記配列番号３の塩基配列における四角で囲んだ塩基配列である。つまり、例えば、前述の第１ＳＮＰセットがうどんこ病抵抗性を示す塩基の組合せを含み、且つ前記配列番号３の塩基配列と対応する塩基配列において、前記配列番号７の塩基配列を含む場合、メロン植物は、うどんこ病抵抗性であり、前記配列番号７の塩基配列を含まない場合、メロン植物は、うどんこ病罹病性であることを示す。なお、前記配列番号７の塩基配列とうどんこ病抵抗性との関連性は、これまでに報告されておらず、本発明者らにより初めて見出された、うどんこ病抵抗性に関与する新規の塩基配列である。 In the condition (2), the resistance locus is further specified by, for example, the 204th to 212th and 213th base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in the base sequence of SEQ ID NO: 3. May be. The base sequence of SEQ ID NO: 7 is a base sequence surrounded by a square in the base sequence of SEQ ID NO: 3. That is, for example, when the first SNP set includes a combination of bases exhibiting resistance to powdery mildew and the base sequence corresponding to the base sequence of SEQ ID NO: 3 includes the base sequence of SEQ ID NO: 7, The plant is resistant to powdery mildew, and when it does not contain the nucleotide sequence of SEQ ID NO: 7, it indicates that the melon plant is susceptible to powdery mildew. The relationship between the nucleotide sequence of SEQ ID NO: 7 and powdery mildew resistance has not been reported so far, and was first discovered by the present inventors and related to the powdery mildew resistance. Is the base sequence.
　本発明において、前記抵抗性遺伝子座は、例えば、前記条件（１）および（２）の一方の条件を満たしてもよいし、両条件を満たしてもよく、うどんこ病抵抗性との相関性がより高いことから、両条件を満たすことが好ましい。 In the present invention, the resistance locus may satisfy, for example, one of the conditions (1) and (2), or may satisfy both conditions, and correlation with powdery mildew resistance. Therefore, it is preferable to satisfy both conditions.
　前記抵抗性遺伝子座は、例えば、前記ＳＮＰの組合せに基づく条件として、さらに、下記条件（３）および（４）の少なくとも一方の条件を満たしてもよい。 The resistance locus may further satisfy at least one of the following conditions (3) and (4) as a condition based on the combination of the SNPs, for example.
条件（３）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号４の塩基配列における５０、１４１、および２６６番目の塩基の多型で特定される。 Condition (3)
The powdery mildew resistance locus on the sixth chromosome is specified by polymorphisms of the 50th, 141st and 266th bases in the base sequence of SEQ ID NO: 4.
Figure JPOXMLDOC01-appb-I000002
Figure JPOXMLDOC01-appb-I000002
条件（４）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号５の塩基配列における９９、１７４、１８４、１９９、および２００番目の塩基の多型で特定される。 Condition (4)
The powdery mildew resistance locus on the sixth chromosome is identified by polymorphisms of the 99th, 174th, 184th, 199th and 200th bases in the base sequence of SEQ ID NO: 5.
配列番号５
5’-GTTCGGATCGGAAAATTCAATCAAAGGAATAAGCGCTACAAAACAATAAACACACACATACATCCAAAAATTACAAATCTCCTATCATCATCAAGAAA[C]AGAAAAACCAAACCGAAAACGAAATAAACGCCAAATAATTTCAGAAAATCGATGCGACGGAATAAGAAATGCAT[G]TATCTGGTA[T]GATCGAAAGAGAAA[A][T]ATACGAGATCCGGTGGTTTGCCAGAGCTGCATCTCGCCGTCTTCATAATCGCCTTCGGGACGAACGGCGATGAGAACAAGGTAATCGCCCTTGCGGACAACGTTGTCGATGGCCCATTTGAGGGCTTTAATGCTGCAGGCAGAGAAATCCACCGCGACGCCGACTCTCCGTTGACCGTCCATGCTTTTTGTGGGTTGGGATTTTCAGTGCGTTTGAGCTTTGTGGAAGGAAGGAAGAATGGAATGAATGTCGGAAAGCTTGG-3’ SEQ ID NO: 5
5'-GTTCGGATCGGAAAATTCAATCAAAGGAATAAGCGCTACAAAACAATAAACACACACATACATCCAAAAATTACAAATCTCCTATCATCATCAAGAAA [C] AGAAAAACCAAACCGAAAACGAAATAAACGCCAAATAATTTCAGAAAATCGATGCGACGGAATAAGAAATGCAT [ G] TATCTGGTA [T] GATCGAAAGAGAAA [A] [T] ATACGAGATCCGGTGGTTTGCCAGAGCTGCATCTCGCCGTCTTCATAATCGCCTTCGGGACGAACGGCGATGAGAACAAGGTAATCGCCCTTGCGGACAACGTTGTCGATGGCCCATTTGAGGGCTTTAATGCTGCAGGCAGAGAAATCCACCGCGACGCCGACTCTCCGTTGACCGTCCATGCTTTTTGTGGGTTGGGATTTTCAGTGCGTTTGAGCTTTGTGGAAGGAAGGAAGAATGGAATGAATGTCGGAAAGCTTGG-3 '
　前記条件（３）において、前記抵抗性遺伝子座は、例えば、前記配列番号４の塩基配列における３種類のＳＮＰの組合せ（以下、「第２ＳＮＰセット」ともいう。）により特定される。前記３種類のＳＮＰは、例えば、前記配列番号４の塩基配列における５０、１４１、および２６６番目のＳＮＰである。以下、５０、１４１、および２６６番目のＳＮＰを、それぞれ、「ＳＮＰ２５０」、「ＳＮＰ２１４１」、および「ＳＮＰ２２６６」ともいう。前記ＳＮＰ２５０、前記ＳＮＰ２１４１、および前記ＳＮＰ２２６６は、それぞれ、前記配列番号４の塩基配列におけるかっこで囲んだ下線部の１、２、および３番目の塩基である。前記配列番号４の塩基配列は、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。前記配列番号４の塩基配列は、例えば、前述のメロン植物のゲノムの塩基配列情報において、第６染色体上の６３９８１３１～６３９８６２７番目の塩基配列に対応する塩基配列である。 In the condition (3), the resistance locus is specified by, for example, a combination of three types of SNPs in the base sequence of SEQ ID NO: 4 (hereinafter also referred to as “second SNP set”). The three types of SNPs are, for example, the 50th, 141st, and 266th SNPs in the base sequence of SEQ ID NO: 4. Hereinafter, the 50th, 141st, and 266th SNPs are also referred to as “SNP2 50 ”, “SNP2 141 ”, and “SNP2 266 ”, respectively. The SNP 2 50 , the SNP 2 141 , and the SNP 2 266 are the first, second, and third bases in the underlined portion in parentheses in the base sequence of SEQ ID NO: 4, respectively. The base sequence of SEQ ID NO: 4 can be obtained, for example, from a melon plant deposited under accession number FERM BP-22291 described later. The base sequence of SEQ ID NO: 4 is, for example, a base sequence corresponding to the 6398131 to 6398627th base sequence on chromosome 6 in the genome sequence information of the melon plant described above.
　前記第２ＳＮＰセットは、例えば、前記配列番号４のかっこで囲んだ下線部の塩基において、１、２、および３番目の塩基が、それぞれ、Ｃ、Ａ、およびＴである多型を示す。つまり、例えば、前記第２ＳＮＰセットにおける前記ＳＮＰ２５０、前記ＳＮＰ２１４１、および前記ＳＮＰ２２６６が、それぞれ、Ｃ、Ａ、およびＴの場合、メロン植物は、うどんこ病抵抗性であり、前記塩基の組合せ以外の塩基の組合せの場合（例えば、Ｔ、Ｔ、およびＡの場合）、メロン植物は、うどんこ病罹病性であることを示す。なお、前記第２ＳＮＰセットとうどんこ病抵抗性との関連性は、これまでに報告されておらず、本発明者らにより初めて見出された、うどんこ病抵抗性に関与する新規のＳＮＰの組合せである。 The second SNP set indicates, for example, polymorphisms in which the first, second, and third bases are C, A, and T, respectively, in the underlined bases surrounded by the brackets of SEQ ID NO: 4. That is, for example, when the SNP2 50 , the SNP2 141 , and the SNP2 266 in the second SNP set are C, A, and T, respectively, the melon plant is resistant to powdery mildew, and the combination of the bases For combinations of bases other than (eg, for T, T, and A), the melon plant is susceptible to powdery mildew. In addition, the relationship between the second SNP set and powdery mildew resistance has not been reported so far, and a novel SNP related to powdery mildew resistance, which was first discovered by the present inventors, was found. It is a combination.
　前記条件（３）において、前記抵抗性遺伝子座は、例えば、さらに、前記配列番号４の塩基配列における１３２および４５７番目のＳＮＰの組合せにより特定されてもよい。以下、１３２および４５７番目のＳＮＰを、それぞれ、「ＳＮＰ２１３２」、および「ＳＮＰ２４５７」ともいう。前記ＳＮＰ２１３２および前記ＳＮＰ２４５７は、それぞれ、前記配列番号４の塩基配列における四角で囲んだ１および２番目の塩基である。 In the condition (3), the resistance locus may be further specified, for example, by a combination of the 132nd and 457th SNPs in the base sequence of SEQ ID NO: 4. Hereinafter, the 132nd and 457th SNPs are also referred to as “SNP2 132 ” and “SNP2 457 ”, respectively. The SNP2 132 and the SNP2 457 are the first and second bases surrounded by a square in the base sequence of SEQ ID NO: 4, respectively.
　前記ＳＮＰ２１３２および前記ＳＮＰ２４５７は、例えば、前記配列番号４の四角で囲んだ下線部の塩基において、１および２番目の塩基が、それぞれ、ＴおよびＡである多型を示す。つまり、例えば、前述の第２ＳＮＰセットがうどんこ病抵抗性を示す塩基の組合せを含み、且つ前記ＳＮＰ２１３２および前記ＳＮＰ２４５７が、それぞれ、ＴおよびＡの場合、メロン植物は、うどんこ病抵抗性であり、前記塩基の組合せ以外の塩基の組合せの場合、メロン植物は、うどんこ病罹病性であることを示す。なお、前記ＳＮＰ２１３２および前記ＳＮＰ２４５７とうどんこ病抵抗性との関連性は、これまでに報告されておらず、本発明者らにより初めて見出された、うどんこ病抵抗性に関与する新規のＳＮＰの組合せである。 The SNP2 132 and the SNP2 457 represent polymorphisms in which the first and second bases are T and A, respectively, in the underlined base surrounded by the square of the SEQ ID NO: 4. That is, for example, when the above-described second SNP set includes a combination of bases exhibiting powdery mildew resistance, and the SNP2 132 and the SNP2 457 are T and A, respectively, the melon plant has the powdery mildew resistance. In the case of a combination of bases other than the combination of bases, it indicates that the melon plant is susceptible to powdery mildew. In addition, the relationship between the SNP2 132 and the SNP2 457 and powdery mildew resistance has not been reported so far, and was first discovered by the present inventors and related to the powdery mildew resistance. Of SNPs.
　前記条件（４）において、前記抵抗性遺伝子座は、例えば、前記配列番号５の塩基配列における５種類のＳＮＰの組合せ（以下、「第３ＳＮＰセット」ともいう。）により特定される。前記５種類のＳＮＰは、例えば、前記配列番号５の塩基配列における９９、１７４、１８４、１９９、および２００番目のＳＮＰである。以下、９９、１７４、１８４、１９９、および２００番目のＳＮＰを、それぞれ、「ＳＮＰ３９９」、「ＳＮＰ３１７４」、「ＳＮＰ３１８４」、「ＳＮＰ３１９９」、および「ＳＮＰ３２００」ともいう。前記ＳＮＰ３９９、前記ＳＮＰ３１７４、前記ＳＮＰ３１８４、前記ＳＮＰ３１９９、および前記ＳＮＰ３２００は、それぞれ、前記配列番号５の塩基配列におけるかっこで囲んだ下線部の１～４および５番目の塩基である。前記配列番号５の塩基配列は、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。前記配列番号５の塩基配列は、例えば、前述のメロン植物のゲノムの塩基配列情報において、第６染色体上の５２６９６４２～５２７０１０３番目の塩基配列に対応する塩基配列である。 In the condition (4), the resistance locus is specified by, for example, a combination of five types of SNPs in the base sequence of SEQ ID NO: 5 (hereinafter also referred to as “third SNP set”). The five types of SNPs are, for example, the 99th, 174th, 184th, 199th and 200th SNPs in the base sequence of SEQ ID NO: 5. Hereinafter, the 99th, 174 , 184 , 199 , and 200th SNPs are also referred to as “SNP3 99 ”, “SNP3 174 ”, “SNP3 184 ”, “SNP3 199 ”, and “SNP3 200 ”, respectively. The SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 are the 1st to 4th and 5th bases in the underlined part in the base sequence of the SEQ ID NO: 5, respectively. The base sequence of SEQ ID NO: 5 can be obtained, for example, from a melon plant deposited under accession number FERM BP-22291 described later. The base sequence of SEQ ID NO: 5 is, for example, the base sequence corresponding to the 5269642-5270103 base sequence on chromosome 6 in the genome sequence information of the melon plant described above.
　前記第３ＳＮＰセットは、例えば、前記配列番号５のかっこで囲んだ下線部の塩基において、１～４および５番目の塩基が、それぞれ、Ｃ、Ｇ、Ｔ、Ａ、およびＴである多型を示す。つまり、例えば、前記第３ＳＮＰセットにおける前記ＳＮＰ３９９、前記ＳＮＰ３１７４、前記ＳＮＰ３１８４、前記ＳＮＰ３１９９、および前記ＳＮＰ３２００が、それぞれ、Ｃ、Ｇ、Ｔ、Ａ、およびＴの場合、メロン植物は、うどんこ病抵抗性であり、前記塩基の組合せ以外の塩基の組合せの場合（例えば、Ｔ、Ａ、Ｃ、Ｔ、およびＣの場合）、メロン植物は、うどんこ病罹病性であることを示す。なお、前記第３ＳＮＰセットとうどんこ病抵抗性との関連性は、これまでに報告されておらず、本発明者らにより初めて見出された、うどんこ病抵抗性に関与する新規のＳＮＰの組合せである。 The third SNP set includes, for example, polymorphisms in which the first to fourth and fifth bases are C, G, T, A, and T, respectively, in the underlined bases enclosed in parentheses of SEQ ID NO: 5. Show. That is, for example, when the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 in the third SNP set are C, G, T, A, and T, respectively, In the case of a combination of bases other than the above-mentioned base combinations that are resistant to powdery mildew (for example, in the case of T, A, C, T, and C), it indicates that the melon plant is susceptible to powdery mildew. . In addition, the relationship between the third SNP set and powdery mildew resistance has not been reported so far, and a novel SNP related to powdery mildew resistance, which was first discovered by the present inventors, was found. It is a combination.
　前記抵抗性遺伝子座が、さらに、前記条件（３）および（４）の少なくとも一方の条件を満たす場合、前記抵抗性遺伝子座が満たす条件の組合せは、特に制限されず、例えば、以下の組合せが例示できる。
条件（１）および（３）
条件（１）および（４）
条件（２）および（３）
条件（２）および（４）
条件（１）、（２）、および（３）
条件（１）、（２）、および（４）
条件（１）、（２）、（３）および（４） When the resistance locus further satisfies at least one of the conditions (3) and (4), the combination of the conditions satisfied by the resistance locus is not particularly limited. For example, the following combinations It can be illustrated.
Conditions (1) and (3)
Conditions (1) and (4)
Conditions (2) and (3)
Conditions (2) and (4)
Conditions (1), (2), and (3)
Conditions (1), (2), and (4)
Conditions (1), (2), (3) and (4)
　なお、本発明において、前記抵抗性遺伝子座は、前記条件（１）および（２）の少なくとも一方の条件を満たすが、前記抵抗性遺伝子座はこれに限定されず、前記条件（１）および（２）の少なくとも一方の条件に代えて、前記条件（３）および（４）の少なくとも一方の条件を満たしてもよい。この場合、前記抵抗性遺伝子座は、例えば、前記条件（３）および（４）の一方の条件を満たしてもよいし、両条件を満たしてもよく、うどんこ病抵抗性との相関性がより高いことから、両条件を満たすことが好ましい。 In the present invention, the resistance locus satisfies at least one of the conditions (1) and (2), but the resistance locus is not limited thereto, and the conditions (1) and ( Instead of at least one of the conditions of 2), at least one of the conditions (3) and (4) may be satisfied. In this case, for example, the resistance locus may satisfy one of the conditions (3) and (4), or may satisfy both conditions, and has a correlation with powdery mildew resistance. Since it is higher, it is preferable to satisfy both conditions.
（ii）塩基配列に基づく条件
　前記抵抗性遺伝子座は、前記（ii）に示すように、例えば、プライマーセットにより増幅される増幅断片の塩基配列およびＳＮＰの組合せを含む塩基配列の少なくとも一方に基づく条件を満たしてもよく、具体的には、下記条件（５）および（６）の少なくとも一方の条件を満たしてもよい。下記条件（５）において、下記（ａ２）および（ａ３）は、それぞれ、前記抵抗性遺伝子座において、前記うどんこ病抵抗性に関して下記（ａ１）と同等の機能を有するポリヌクレオチドである。下記条件（６）において、下記（ｂ２）および（ｂ３）は、それぞれ、前記抵抗性遺伝子座において、前記うどんこ病抵抗性に関して下記（ｂ１）と同等の機能を有するポリヌクレオチドである。 (Ii) Conditions based on nucleotide sequence As shown in (ii) above, the resistance locus is based on, for example, at least one of the nucleotide sequence of an amplified fragment amplified by a primer set and a nucleotide sequence including a combination of SNPs. The condition may be satisfied. Specifically, at least one of the following conditions (5) and (6) may be satisfied. In the following condition (5), the following (a2) and (a3) are polynucleotides having a function equivalent to that of the following (a1) regarding the powdery mildew resistance at the resistance locus. In the following condition (6), the following (b2) and (b3) are polynucleotides having functions equivalent to those in the following (b1) with respect to the powdery mildew resistance at the resistance locus.
条件（５）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ａ）のポリヌクレオチドで特定される。
（ａ）　下記（ａ１）、（ａ２）、または（ａ３）のポリヌクレオチド
（ａ１）配列番号６の塩基配列からなるポリヌクレオチド
（ａ２）前記（ａ１）において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなるポリヌクレオチド
（ａ３）前記（ａ１）の塩基配列に対して、８０％以上の同一性を有する塩基配列からなるポリヌクレオチド Condition (5)
The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (a) below.
(A) polynucleotide (a2) comprising the nucleotide sequence of SEQ ID NO: 6 (a1), polynucleotide (a1) below (a1), (a2), or (a3): one or several bases deleted in (a1) A polynucleotide comprising a base sequence that has been substituted, inserted and / or added (a3) a polynucleotide comprising a base sequence having 80% or more identity to the base sequence of (a1) above
条件（６）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｂ）のポリヌクレオチドで特定される。
（ｂ）　下記（ｂ１）、（ｂ２）、または（ｂ３）のポリヌクレオチド
（ｂ１）配列番号３の塩基配列からなるポリヌクレオチド
（ｂ２）前記（ｂ１）の４５番目の塩基（Ａ）、４８番目の塩基（Ａ）、４９番目の塩基（Ｔ）、５１番目の塩基（Ｔ）、１０８番目の塩基（Ｔ）、１２０番目の塩基（Ａ）、１３９番目の塩基（Ａ）、２１４番目の塩基（Ｔ）、および３２７番目の塩基（Ｃ）が保存され、前記塩基以外の塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなるポリヌクレオチド
（ｂ３）前記（ｂ１）の４５番目の塩基（Ａ）、４８番目の塩基（Ａ）、４９番目の塩基（Ｔ）、５１番目の塩基（Ｔ）、１０８番目の塩基（Ｔ）、１２０番目の塩基（Ａ）、１３９番目の塩基（Ａ）、２１４番目の塩基（Ｔ）、および３２７番目の塩基（Ｃ）が保存され、前記塩基以外の塩基配列に対して、８０％以上の同一性を有する塩基配列からなるポリヌクレオチド Condition (6)
The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (b) below.
(B) Polynucleotide (b2) of the following (b1), (b2), or (b3) (b1) SEQ ID NO: 3 (b2) 45th base (A), 48th of (b1) Base (A), 49th base (T), 51st base (T), 108th base (T), 120th base (A), 139th base (A), 214th base (T) and a polynucleotide comprising a base sequence in which the 327th base (C) is conserved and one or several bases are deleted, substituted, inserted and / or added in a base sequence other than the base ( b3) 45th base (A), 48th base (A), 49th base (T), 51st base (T), 108th base (T), 120th base of (b1) Base (A), 139th base (A) , 214th base (T), and 327th base (C), a polynucleotide comprising a base sequence having 80% or more identity to a base sequence other than the base
　前記（ａ１）のポリヌクレオチドにおいて、配列番号６の塩基配列は、以下の通りである。前記（ａ１）のポリヌクレオチド（配列番号６の塩基配列）は、例えば、前記プライマーセット１を用いて、例えば、メロン植物について遺伝子増幅を行うことで得られる。前記配列番号６の塩基配列は、例えば、前記増幅断片において、前記フォワードプライマー１を含む増幅断片の塩基配列である。また、前記（ａ１）のポリヌクレオチドは、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。
配列番号６
5’-AATCTCAACAAGTGAGCTTTTATTGTAAAAAATACAACACAAGTAAGAGTGTGTGTATTTATAATTGAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAAACAAGACAAAAAGAAAATTGAAGATAATCATG-3’ In the polynucleotide (a1), the base sequence of SEQ ID NO: 6 is as follows. The polynucleotide (a1) (base sequence of SEQ ID NO: 6) can be obtained, for example, by performing gene amplification on, for example, a melon plant using the primer set 1. The base sequence of SEQ ID NO: 6 is, for example, the base sequence of the amplified fragment containing the forward primer 1 in the amplified fragment. The polynucleotide (a1) can be obtained, for example, from a melon plant deposited under the deposit number FERM BP-22291 described later.
SEQ ID NO: 6
5'-AATCTCAACAAGTGAGCTTTTATTGTAAAAAATACAACACAAGTAAGAGTGTGTGTATTTATAATTGAAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAAACAAGACAAAAAGAAAATTGAAGATAATCATG-3 '
　前記（ａ２）のポリヌクレオチドにおいて、前記「１もしくは数個」は、例えば、１～２７個、１～２０個、１～１５個、１～７個、１～５個、１～４個、１～３個、１個または２個である。本発明において、塩基数等の個数の数値範囲は、例えば、その範囲に属する正の整数を全て開示するものである。つまり、例えば、「１～５個」との記載は、「１、２、３、４、５個」の全ての開示を意味する（以下、同様）。 In the polynucleotide (a2), the “one or several” is, for example, 1 to 27, 1 to 20, 1 to 15, 1 to 7, 1 to 5, 1 to 4, One to three, one or two. In the present invention, the numerical range of the number of bases and the like discloses, for example, all positive integers belonging to the range. That is, for example, the description “1 to 5” means all disclosures of “1, 2, 3, 4, 5” (hereinafter the same).
　前記（ａ３）のポリヌクレオチドにおいて、前記「同一性」は、例えば、８０％以上、８５％以上、８９％以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。前記「同一性」は、２つの塩基配列をアライメントすることによって求めることができる（以下、同様）。 In the polynucleotide (a3), the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. The “identity” can be determined by aligning two base sequences (hereinafter the same).
　前記（ｂ１）のポリヌクレオチドにおいて、配列番号３におけるかっこで囲んだ下線部の１～８および９番目の塩基が、前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７の多型に対応する塩基である。前記（ｂ１）のポリヌクレオチドは、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。 In the polynucleotide (b1), the underlined first to eighth and ninth bases surrounded by parentheses in SEQ ID NO: 3 are the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , It is a base corresponding to the polymorphisms of SNP1 120 , SNP1 139 , SNP1 214 , and SNP1 327 . The polynucleotide (b1) can be obtained, for example, from a melon plant deposited under the accession number FERM BP-22291 described later.
　前記（ｂ２）のポリヌクレオチドにおいて、前記「１もしくは数個」は、例えば、１～１１２個、１～８４個、１～６２個、１～５６個、１～２８個、１～２３個、１～１７個、１～１２個、１～６個、１～３個、１個または２個である。 In the polynucleotide (b2), the “one or several” is, for example, 1 to 112, 1 to 84, 1 to 62, 1 to 56, 1 to 28, 1 to 23, 1 to 17, 1 to 12, 1 to 6, 1 to 3, 1, or 2.
　前記（ｂ２）のポリヌクレオチドは、例えば、さらに、前記（ｂ１）における２０４～２１２および２１３番目の塩基配列（配列番号７：5’-AAAAGCTCCA-3’）が保存されてもよい。この場合、前記（ｂ２）のポリヌクレオチドは、例えば、前記（ｂ１）における４５番目の塩基（Ａ）、４８番目の塩基（Ａ）、４９番目の塩基（Ｔ）、５１番目の塩基（Ｔ）、１０８番目の塩基（Ｔ）、１２０番目の塩基（Ａ）、１３９番目の塩基（Ａ）、２１４番目の塩基（Ｔ）、および３２７番目の塩基（Ｃ）、ならびに２０４～２１２および２１３番目の塩基配列（5’-AAAAGCTCCA-3’）が保存され、前記塩基以外の塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなるポリヌクレオチドである。 In the polynucleotide (b2), for example, the 204th to 212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1) above may be further preserved. In this case, the polynucleotide of (b2) is, for example, the 45th base (A), 48th base (A), 49th base (T), 51st base (T) in (b1). , 108th base (T), 120th base (A), 139th base (A), 214th base (T), and 327th base (C), and 204-212 and 213th A polynucleotide comprising a nucleotide sequence in which a nucleotide sequence (5'-AAAAGCTCCA-3 ') is conserved and one or several bases are deleted, substituted, inserted and / or added in a nucleotide sequence other than the aforementioned nucleotide sequence .
　前記（ｂ３）のポリヌクレオチドにおいて、前記「同一性」は、例えば、８０％以上、８５％以上、８９％以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。 In the polynucleotide (b3), the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
　前記（ｂ３）のポリヌクレオチドは、例えば、さらに、前記（ｂ１）における２０４～２１２および２１３番目の塩基配列（配列番号７：5’-AAAAGCTCCA-3’）が保存されてもよい。この場合、前記（ｂ３）のポリヌクレオチドは、例えば、前記（ｂ１）における４５番目の塩基（Ａ）、４８番目の塩基（Ａ）、４９番目の塩基（Ｔ）、５１番目の塩基（Ｔ）、１０８番目の塩基（Ｔ）、１２０番目の塩基（Ａ）、１３９番目の塩基（Ａ）、２１４番目の塩基（Ｔ）、および３２７番目の塩基（Ｃ）、ならびに２０４～２１２および２１３番目の塩基配列（5’-AAAAGCTCCA-3’）が保存され、前記塩基以外の塩基配列に対して、８０％以上の同一性を有する塩基配列からなるポリヌクレオチドである。 In the polynucleotide (b3), for example, the 204th to 212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1) above may be further preserved. In this case, the polynucleotide of (b3) is, for example, 45th base (A), 48th base (A), 49th base (T), 51st base (T) in (b1). , 108th base (T), 120th base (A), 139th base (A), 214th base (T), and 327th base (C), and 204-212 and 213th A polynucleotide comprising a nucleotide sequence having a nucleotide sequence (5′-AAAAGCTCCA-3 ′) conserved and having 80% or more identity to a nucleotide sequence other than the aforementioned nucleotide sequence.
　前記抵抗性遺伝子座が前記条件（５）および（６）の少なくとも一方の条件を満たす場合、前記抵抗性遺伝子座は、例えば、前記条件（５）および（６）の一方の条件を満たしてもよいし、両条件を満たしてもよく、うどんこ病抵抗性との相関性がより高いことから、両条件を満たすことが好ましい。 When the resistance locus satisfies at least one of the conditions (5) and (6), the resistance locus may satisfy, for example, one of the conditions (5) and (6). Both conditions may be satisfied, and it is preferable to satisfy both conditions because of higher correlation with powdery mildew resistance.
　前記抵抗性遺伝子座は、例えば、前記塩基配列に基づく条件として、さらに、下記条件（７）および（８）の少なくとも一方の条件を満たしてもよい。下記条件（７）において、下記（ｃ２）および（ｃ３）は、それぞれ、前記抵抗性遺伝子座において、前記うどんこ病抵抗性に関して下記（ｃ１）と同等の機能を有するポリヌクレオチドである。下記条件（８）において、下記（ｄ２）および（ｄ３）は、それぞれ、前記抵抗性遺伝子座において、前記うどんこ病抵抗性に関して下記（ｄ１）と同等の機能を有するポリヌクレオチドである。 The resistance locus may satisfy, for example, at least one of the following conditions (7) and (8) as a condition based on the base sequence. In the following condition (7), the following (c2) and (c3) are polynucleotides having functions equivalent to those in the following (c1) with respect to the powdery mildew resistance at the resistance locus. In the following condition (8), the following (d2) and (d3) are polynucleotides having a function equivalent to that of the following (d1) with respect to the powdery mildew resistance at the resistance locus.
条件（７）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｃ）のポリヌクレオチドで特定される。
（ｃ）　下記（ｃ１）、（ｃ２）、または（ｃ３）のポリヌクレオチド
（ｃ１）配列番号４の塩基配列からなるポリヌクレオチド
（ｃ２）前記（ｃ１）の５０番目の塩基（Ｃ）、１４１番目の塩基（Ａ）、および２６６番目の塩基（Ｔ）が保存され、前記塩基以外の塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなるポリヌクレオチド
（ｃ３）前記（ｃ１）の５０番目の塩基（Ｃ）、１４１番目の塩基（Ａ）、および２６６番目の塩基（Ｔ）が保存され、前記塩基以外の塩基配列に対して、８０％以上の同一性を有する塩基配列からなるポリヌクレオチド Condition (7)
The powdery mildew resistance locus on chromosome 6 is specified by the polynucleotide (c) below.
(C) Polynucleotide (c2) consisting of the base sequence of SEQ ID NO: 4 (c1), (c1), (c2), or (c3) below The 50th base (C), 141st of (c1) Of the base (A) and the 266th base (T), and a base sequence other than the base, wherein one or several bases are deleted, substituted, inserted and / or added. Nucleotide (c3) The 50th base (C), 141st base (A), and 266th base (T) of (c1) are stored, and 80% or more of the base sequence other than the base A polynucleotide comprising a nucleotide sequence having the same identity
条件（８）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｄ）のポリヌクレオチドで特定される。
（ｄ）　下記（ｄ１）、（ｄ２）、または（ｄ３）のポリヌクレオチド
（ｄ１）配列番号５の塩基配列からなるポリヌクレオチド
（ｄ２）前記（ｄ１）の９９番目の塩基（Ｃ）、１７４番目の塩基（Ｇ）、１８４番目の塩基（Ｔ）、１９９番目の塩基（Ａ）、および２００番目の塩基（Ｔ）が保存され、前記塩基以外の塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなるポリヌクレオチド
（ｄ３）前記（ｄ１）の９９番目の塩基（Ｃ）、１７４番目の塩基（Ｇ）、１８４番目の塩基（Ｔ）、１９９番目の塩基（Ａ）、および２００番目の塩基（Ｔ）が保存され、前記塩基以外の塩基配列に対して、８０％以上の同一性を有する塩基配列からなるポリヌクレオチド Condition (8)
The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (d) below.
(D) polynucleotide (d2) comprising the nucleotide sequence of SEQ ID NO: 5 (d1), polynucleotide (d1) below (d1), (d2) or (d3) 99th base (C), 174th of (d1) Base (G), 184th base (T), 199th base (A), and 200th base (T) are conserved, and in the base sequence other than the base, one or several bases are missing. Polynucleotide (d3) comprising the nucleotide sequence deleted, substituted, inserted and / or added (d3) 99th base (C), (174) base (G), 184th base (T), 199 A polynucleotide comprising a base sequence having 80% or more identity to a base sequence other than the base, wherein the base (A) and the base 200 (T) are conserved
　前記（ｃ１）のポリヌクレオチドにおいて、配列番号４におけるかっこで囲んだ下線部の１、２、および３番目の塩基が、前記ＳＮＰ２５０、前記ＳＮＰ２１４１、および前記ＳＮＰ２２６６の多型に対応する塩基である。前記（ｃ１）のポリヌクレオチドは、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。 In the polynucleotide (c1), the first, second, and third bases in the underlined brackets in SEQ ID NO: 4 are bases corresponding to the polymorphisms of the SNP2 50 , the SNP2 141 , and the SNP2 266 It is. The polynucleotide (c1) can be obtained, for example, from a melon plant deposited under the deposit number FERM BP-22291 described later.
　前記（ｃ２）のポリヌクレオチドにおいて、前記「１もしくは数個」は、例えば、１～１００、１～７５個、１～５５個、１～５０個、１～２５個、１～２０個、１～１５個、１～１０個、１～５個、１～３個、１個または２個である。 In the polynucleotide (c2), the “one or several” is, for example, 1 to 100, 1 to 75, 1 to 55, 1 to 50, 1 to 25, 1 to 20, -15, 1-10, 1-5, 1-3, 1 or 2.
　前記（ｃ２）のポリヌクレオチドは、例えば、さらに、前記（ｃ１）における１３２番目の塩基（Ｔ）および４５７番目の塩基（Ａ）が保存されてもよい。この場合、前記（ｃ２）のポリヌクレオチドは、例えば、前記（ｃ１）における５０番目の塩基（Ｃ）、１３２番目の塩基（Ｔ）、１４１番目の塩基（Ａ）、２６６番目の塩基（Ｔ）、および４５７番目の塩基（Ａ）が保存され、前記塩基以外の塩基配列において、１もしくは数個の塩基が欠失、置換、挿入および／または付加された塩基配列からなるポリヌクレオチドである。 In the polynucleotide (c2), for example, the 132nd base (T) and the 457th base (A) in (c1) may be further preserved. In this case, the polynucleotide (c2) is, for example, the 50th base (C), the 132nd base (T), the 141st base (A), and the 266th base (T) in (c1). 457, and the 457th base (A), and is a polynucleotide comprising a base sequence in which one or several bases are deleted, substituted, inserted and / or added in a base sequence other than the base.
　前記（ｃ３）のポリヌクレオチドにおいて、前記「同一性」は、例えば、８０％以上、８５％以上、８９％以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。 In the polynucleotide (c3), the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
　前記（ｃ３）のポリヌクレオチドは、例えば、さらに、前記（ｃ１）における１３２番目の塩基（Ｔ）および４５７番目の塩基（Ａ）が保存されてもよい。この場合、前記（ｃ３）のポリヌクレオチドは、例えば、前記（ｃ１）における５０番目の塩基（Ｃ）、１３２番目の塩基（Ｔ）、１４１番目の塩基（Ａ）、２６６番目の塩基（Ｔ）、および４５７番目の塩基（Ａ）が保存され、前記塩基以外の塩基配列に対して、８０％以上の同一性を有する塩基配列からなるポリヌクレオチドである。 In the polynucleotide (c3), for example, the 132nd base (T) and the 457th base (A) in (c1) may be further preserved. In this case, the polynucleotide of (c3) is, for example, the 50th base (C), the 132nd base (T), the 141st base (A), and the 266th base (T) in (c1). 457 and the 457th base (A), and is a polynucleotide comprising a base sequence having 80% or more identity to a base sequence other than the base.
　前記（ｄ１）のポリヌクレオチドにおいて、配列番号５におけるかっこで囲んだ下線部の１～４および５番目の塩基が、前記ＳＮＰ３９９、前記ＳＮＰ３１７４、前記ＳＮＰ３１８４、前記ＳＮＰ３１９９、および前記ＳＮＰ３２００の多型に対応する塩基である。前記（ｄ１）のポリヌクレオチドは、例えば、後述する受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物から得ることができる。 In the polynucleotide of (d1), the first to fourth and fifth bases in the underlined brackets in SEQ ID NO: 5 are the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 It is a base corresponding to the polymorphism of. The polynucleotide (d1) can be obtained, for example, from a melon plant deposited under the deposit number FERM BP-22291 described later.
　前記（ｄ２）のポリヌクレオチドにおいて、前記「１もしくは数個」は、例えば、１～９３、１～７０個、１～５１個、１～４８個、１～２４個、１～１９個、１～１５個、１～１０個、１～５個、１～３個、１個または２個である。 In the polynucleotide (d2), the “one or several” is, for example, 1 to 93, 1 to 70, 1 to 51, 1 to 48, 1 to 24, 1 to 19, -15, 1-10, 1-5, 1-3, 1 or 2.
　前記（ｄ３）のポリヌクレオチドにおいて、前記「同一性」は、例えば、８０％以上、８５％以上、８９％以上、９０％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。 In the polynucleotide (d3), the “identity” is, for example, 80% or more, 85% or more, 89% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more.
　前記抵抗性遺伝子座が、さらに、前記条件（７）および（８）の少なくとも一方の条件を満たす場合、前記抵抗性遺伝子座が満たす条件の組合せは、特に制限されず、例えば、以下の組合せが例示できる。
条件（５）および（７）
条件（５）および（８）
条件（６）および（７）
条件（６）および（８）
条件（５）、（６）、および（７）
条件（５）、（６）、および（８）
条件（５）、（６）、（７）および（８） When the resistance locus further satisfies at least one of the conditions (7) and (8), the combination of the conditions satisfied by the resistance locus is not particularly limited. For example, the following combinations It can be illustrated.
Conditions (5) and (7)
Conditions (5) and (8)
Conditions (6) and (7)
Conditions (6) and (8)
Conditions (5), (6), and (7)
Conditions (5), (6), and (8)
Conditions (5), (6), (7) and (8)
　なお、本発明において、前記抵抗性遺伝子座が、前記（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件を満たすが、前記抵抗性遺伝子座はこれに限定されず、前述のように、前記（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件に代えて、前記（ii）塩基配列に基づく条件を満たしてもよい。この場合、前記抵抗性遺伝子座は、例えば、前記条件（５）および（６）の少なくとも一方の条件に代えて、前記条件（７）および（８）の少なくとも一方の条件を満たしてもよい。また、この場合、前記抵抗性遺伝子座は、例えば、前記条件（７）および（８）の一方の条件を満たしてもよいし、両条件を満たしてもよく、うどんこ病抵抗性との相関性がより高いことから、両条件を満たすことが好ましい。 In the present invention, the resistance locus satisfies the condition based on the combination of the length (i) of the amplified fragment and the SNP, but the resistance locus is not limited to this, and as described above, Instead of the condition (i) based on the combination of the length of the amplified fragment and the SNP, the condition (ii) based on the base sequence may be satisfied. In this case, for example, the resistance locus may satisfy at least one of the conditions (7) and (8) instead of at least one of the conditions (5) and (6). In this case, for example, the resistance locus may satisfy one of the conditions (7) and (8), or may satisfy both conditions, and correlation with powdery mildew resistance. It is preferable that both conditions are satisfied because the property is higher.
（iii）領域の塩基配列に基づく条件
　前記抵抗性遺伝子座は、前記（iii）に示すように、例えば、プライマーセットで増幅される増幅断片の塩基配列およびＳＮＰの組合せを含む塩基配列の少なくとも一方を含む領域の塩基配列に基づく条件を満たしてもよく、具体的には、下記条件（９）を満たしてもよい。前記領域は、例えば、前記増幅断片の塩基配列および前記ＳＮＰの組合せを含む塩基配列の少なくとも一方の塩基配列の全体を含む条件でもよいし、一部を含む条件でもよい。 (Iii) Conditions based on the base sequence of the region As shown in the above (iii), the resistance locus is, for example, at least one of the base sequence of the amplified fragment amplified with the primer set and the base sequence including the combination of SNPs. The condition based on the base sequence of the region including may be satisfied, and specifically, the following condition (9) may be satisfied. The region may be, for example, a condition including the entire base sequence of the amplified fragment and at least one of the base sequences including the combination of the SNPs or a condition including a part thereof.
条件（９）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記染色体における下記（Ａ）、（Ｂ）および（Ｃ）のＳＮＰからなる群から選択された２つのＳＮＰの部位間の塩基配列により特定される。
（Ａ）　配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目のＳＮＰからなる群から選択された１つのＳＮＰ
（Ｂ）　配列番号４の塩基配列における５０、１３２、１４１、２６６、および４５７番目のＳＮＰからなる群から選択された１つのＳＮＰ
（Ｃ）　配列番号５の塩基配列における９９、１７４、１８４、１９９、および２００番目のＳＮＰからなる群から選択された１つのＳＮＰ Condition (9)
The powdery mildew resistance locus on the chromosome 6 is identified by the base sequence between two SNP sites selected from the group consisting of the following SNPs (A), (B) and (C) in the chromosome: Is done.
(A) One SNP selected from the group consisting of 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th SNPs in the base sequence of SEQ ID NO: 3
(B) One SNP selected from the group consisting of 50th, 132, 141, 266, and 457th SNPs in the base sequence of SEQ ID NO: 4
(C) One SNP selected from the group consisting of the 99th, 174, 184, 199, and 200th SNPs in the base sequence of SEQ ID NO: 5
　前記（Ａ）のＳＮＰにおいて、４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目のＳＮＰは、例えば、それぞれ、前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７の多型に対応する塩基である。 In the SNP of (A), the 45th , 48th , 49th , 51st , 108th, 120th, 139th, 214th, and 327th SNPs are, for example, the SNP1 45 , the SNP1 48 , the SNP1 49 , and the SNP1 51, respectively. , SNP1 108 , SNP1 120 , SNP1 139 , SNP1 214 , and SNP1 327 bases corresponding to polymorphisms.
　前記（Ｂ）のＳＮＰにおいて、５０、１３２、１４１、２６６、および４５７番目のＳＮＰは、例えば、それぞれ、前記ＳＮＰ２５０、前記ＳＮＰ２１３２、前記ＳＮＰ２１４１、前記ＳＮＰ２２６６、および前記ＳＮＰ２４５７の多型に対応する塩基である。 In the SNP of (B), the 50 , 132 , 141 , 266 , and 457th SNPs are, for example, polymorphisms of the SNP2 50 , the SNP2 132 , the SNP2 141 , the SNP2 266 , and the SNP2 457 , respectively. Is a base corresponding to
　前記（Ｃ）のＳＮＰにおいて、９９、１７４、１８４、１９９、および２００番目のＳＮＰは、例えば、それぞれ、前記ＳＮＰ３９９、前記ＳＮＰ３１７４、前記ＳＮＰ３１８４、前記ＳＮＰ３１９９、および前記ＳＮＰ３２００の多型に対応する塩基である。 In the SNP of (C), the 99 , 174 , 184 , 199 , and 200th SNPs are, for example, polymorphisms of the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 , respectively. Is a base corresponding to
　前記領域は、前述のように、例えば、（Ａ）、（Ｂ）および（Ｃ）のＳＮＰからなる群から選択された２つのＳＮＰの部位によって、上流側端部と下流側端部とを特定できる。前記領域は、例えば、前記（Ａ）、（Ｂ）および（Ｃ）のＳＮＰからなる群から選択された２つのＳＮＰの部位間であればよく、例えば、前記２つのＳＮＰの部位の両方または一方を含んでもよいし、含まなくてもよい。また、前記領域が、前記ＳＮＰの部位を含む場合、前記領域の前記上流側端部と前記下流側端部とは、前記ＳＮＰの部位となるが、前記上流側端部と前記下流側端部との塩基は、例えば、前述した塩基配列における下線部または四角で囲んだ塩基でもよいし、それ以外の塩基でもよい。 As described above, for example, the region identifies an upstream end and a downstream end by two SNP sites selected from the group consisting of SNPs (A), (B), and (C). it can. The region may be, for example, between two SNP sites selected from the group consisting of the SNPs of (A), (B) and (C), for example, both or one of the two SNP sites. May or may not be included. When the region includes the SNP part, the upstream end and the downstream end of the region are the SNP part, but the upstream end and the downstream end. The base may be, for example, a base surrounded by an underline or a square in the base sequence described above, or may be a base other than that.
　具体的に、前記上流側端部と前記下流側端部とのＳＮＰは、例えば、前記（Ａ）、（Ｂ）、および（Ｃ）のＳＮＰの座乗位置に基づき、決定できる。前記（Ａ）、（Ｂ）、および（Ｃ）のＳＮＰは、例えば、図１に示すように、メロン植物の第６染色体上において、上流側（ＳＮＰ３９９）から下流側（ＳＮＰ２４５７）にかけて、前記（Ｃ）のＳＮＰ、前記（Ａ）のＳＮＰ、および前記（Ｂ）のＳＮＰがこの順で座乗している。また、図１に示すように、前記増幅断片の塩基配列は、例えば、前記（Ａ）のＳＮＰおよび前記（Ｂ）のＳＮＰの間に座乗している。この場合、前記領域を特定する前記２つのＳＮＰは、例えば、以下の組合せが例示できる。
（Ａ）のＳＮＰと（Ｂ）のＳＮＰとの組合せ
（Ａ）のＳＮＰと（Ｃ）のＳＮＰとの組合せ
（Ｂ）のＳＮＰと（Ｃ）のＳＮＰとの組合せ
前記組合せのうち、うどんこ病抵抗性との相関性がより高いことから、好ましくは、例えば、以下の組合せである。
（Ｂ）のＳＮＰと（Ｃ）のＳＮＰとの組合せ Specifically, the SNP between the upstream end and the downstream end can be determined based on the seating positions of the SNPs (A), (B), and (C), for example. The SNPs of (A), (B), and (C) are, for example, from the upstream side (SNP3 99 ) to the downstream side (SNP2 457 ) on chromosome 6 of the melon plant, as shown in FIG. The SNP of (C), the SNP of (A), and the SNP of (B) are seated in this order. Moreover, as shown in FIG. 1, the base sequence of the amplified fragment is located between, for example, the SNP of (A) and the SNP of (B). In this case, examples of the two SNPs that specify the region include the following combinations.
Combination of SNP of (A) and SNP of (B) Combination of SNP of (A) and SNP of (C) Combination of SNP of (B) and SNP of (C) Of the above combinations, powdery mildew Since the correlation with the resistance is higher, for example, the following combinations are preferable.
Combination of (B) SNP and (C) SNP
　前記２つのＳＮＰの部位間の領域の塩基配列によって、前記抵抗性遺伝子座を特定する場合、前記抵抗性遺伝子座は、さらに、前記領域の塩基配列に関連する前述の条件を含むことが好ましい。具体的には、前記抵抗性遺伝子座は、前記領域の塩基配列において、例えば、前記条件（１）および（２）の少なくとも一方の条件を満たすことが好ましい。また、前記抵抗性遺伝子座は、さらに、前記領域の塩基配列において、例えば、前記条件（３）および（４）の少なくとも一方の条件を満たすことが好ましい。 When the resistance locus is specified by the base sequence of the region between the two SNP sites, it is preferable that the resistance locus further includes the above-described conditions related to the base sequence of the region. Specifically, the resistance locus preferably satisfies, for example, at least one of the conditions (1) and (2) in the base sequence of the region. Moreover, it is preferable that the resistance locus further satisfies, for example, at least one of the conditions (3) and (4) in the base sequence of the region.
　前記関連する条件は、例えば、前記領域を特定する上流側端のＳＮＰの部位と前記下流側端のＳＮＰの部位との間の塩基配列に関連する条件があげられ、例えば、前記図１に示す前記（Ａ）、（Ｂ）および（Ｃ）のＳＮＰ、ならびに前記増幅断片の塩基配列の座乗位置に基づき、適宜決定できる。前記関連する条件の数は、例えば、１つ以上であればよく、具体例として、前記領域を特定するＳＮＰの部位間に座乗する塩基配列に関連する全ての条件である。 Examples of the related conditions include conditions related to the base sequence between the SNP site at the upstream end and the SNP site at the downstream end that specify the region, and are shown in FIG. It can be determined appropriately based on the SNPs of (A), (B) and (C) and the locus position of the base sequence of the amplified fragment. The number of the related conditions may be one or more, for example, and as a specific example, is all conditions related to the base sequence seated between the SNP sites specifying the region.
　前記抵抗性遺伝子座が満たす前記２つのＳＮＰの部位間の領域の塩基配列と前記領域の塩基配列に関連する条件との組合せは、特に制限されず、例えば、下記条件（ａ）、（ｂ）または（ｃ）があげられる。
条件（ａ）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記染色体における、（Ａ）のＳＮＰおよび（Ｂ）のＳＮＰの部位間の領域の塩基配列を含み、且つ、
条件（１）および（２）の少なくとも一方の条件を満たす。
条件（ｂ）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記染色体における、（Ｂ）のＳＮＰおよび（Ｃ）のＳＮＰの部位間の領域の塩基配列を含み、且つ、
条件（１）および（２）の少なくとも一方の条件を満たす。
条件（ｃ）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記染色体における、（Ｂ）のＳＮＰおよび（Ｃ）のＳＮＰの部位間の領域の塩基配列を含み、且つ、
条件（１）および（２）の少なくとも一方の条件と条件（３）および（４）の少なくとも一方の条件とを満たす。 The combination of the base sequence of the region between the two SNP sites satisfied by the resistance locus and the conditions related to the base sequence of the region is not particularly limited. For example, the following conditions (a) and (b) Or (c).
Condition (a)
The powdery mildew resistance locus on the chromosome 6 comprises the nucleotide sequence of the region between the sites of the SNP of (A) and the SNP of (B) in the chromosome, and
Satisfy at least one of the conditions (1) and (2).
Condition (b)
The powdery mildew resistance locus on the chromosome 6 comprises the nucleotide sequence of the region between the site of the SNP of (B) and the SNP of (C) in the chromosome, and
Satisfy at least one of the conditions (1) and (2).
Condition (c)
The powdery mildew resistance locus on the chromosome 6 comprises the nucleotide sequence of the region between the site of the SNP of (B) and the SNP of (C) in the chromosome, and
At least one of the conditions (1) and (2) and at least one of the conditions (3) and (4) are satisfied.
　前記条件（ａ）において、前記抵抗性遺伝子座は、例えば、前記条件（１）および（２）の一方の条件を満たしてもよいし、両条件を満たしてもよい。 In the condition (a), the resistance locus may satisfy, for example, one of the conditions (1) and (2), or may satisfy both conditions.
　前記条件（ｂ）において、前記抵抗性遺伝子座は、例えば、前記条件（１）および（２）の一方の条件を満たしてもよいし、両条件を満たしてもよい。 In the condition (b), for example, the resistance locus may satisfy one of the conditions (1) and (2), or may satisfy both conditions.
　前記条件（ｃ）において、前記抵抗性遺伝子座が満たす条件の組合せは、特に制限されず、例えば、以下の組合せが例示できる。
条件（１）および（３）
条件（１）および（４）
条件（２）および（３）
条件（２）および（４）
条件（１）、（２）、および（３）
条件（１）、（２）、および（４）
条件（１）、（２）、（３）および（４） In the condition (c), the combination of conditions satisfied by the resistance locus is not particularly limited, and examples thereof include the following combinations.
Conditions (1) and (3)
Conditions (1) and (4)
Conditions (2) and (3)
Conditions (2) and (4)
Conditions (1), (2), and (3)
Conditions (1), (2), and (4)
Conditions (1), (2), (3) and (4)
　本発明の抵抗性マーカーによれば、例えば、メロン植物に対して、うどんこ病抵抗性を付与することができる。本発明において、メロン植物の前記うどんこ病抵抗性の程度は、例えば、下記参考文献２に記載の方法を参照し、発病指数により表わすことができる。この方法による前記発病指数の算出は、後述する実施例１の説明を援用でき、例えば、発病指数１以下を耐病性（抵抗性）、発病指数２以上を罹病性と設定できる。
参考文献２：LongZhou　Liu　et.al.,　“A　Sequence-amplified　Characterized　Region　Marker　for　a　Single,　Dominant　Gene　in　Melon　PI　134198　that　Confers　Resistance　to　a　Unique　Race　of　Podosphaera　xanthii　in　China”,　HORTSCIENCE,　2010,　vol.45,　No.9,　pp.1407-1410 According to the resistance marker of the present invention, for example, powdery mildew resistance can be imparted to melon plants. In the present invention, the degree of powdery mildew resistance of a melon plant can be represented by, for example, a disease index by referring to the method described in Reference Document 2 below. For the calculation of the disease index by this method, the description of Example 1 described later can be used. For example, a disease index of 1 or less can be set as disease resistance (resistance), and a disease index of 2 or more can be set as susceptibility.
Reference 2: LongZhou Liu et.al., “A Sequence-amplified Characterized Region Marker for a Single, Dominant Gene in Melon PI 134198 that Confers Resistance to a Unique Race of Podosphaera xanthii in China”, HORTSCIENCE, 2010, vol.45 , No.9, pp.1407-1410
　本発明の抵抗性マーカーは、例えば、さらに、他の抵抗性マーカーを含んでもよい。 The resistance marker of the present invention may further include another resistance marker, for example.
２．うどんこ病抵抗性メロン植物
　本発明のうどんこ病抵抗性メロン植物は、前述のように、第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする。
条件（１）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
前記増幅断片の長さが、１３３塩基長以上である。
プライマーセット１
　　　配列番号１の塩基配列からなるフォワードプライマー１
　　　配列番号２の塩基配列からなるリバースプライマー１
条件（２）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。 2. Powdery mildew resistant melon plant The powdery mildew resistant melon plant of the present invention comprises a powdery mildew resistant gene locus on chromosome 6 in a homozygous form as described above,
The powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
Condition (1)
The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
The length of the amplified fragment is 133 bases or more.
Primer set 1
Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
Condition (2)
The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
　本発明のうどんこ病抵抗性メロン植物は、前記第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とし、その他の構成および条件は、特に制限されない。本発明のうどんこ病抵抗性メロン植物は、前記抵抗性遺伝子座である前記本発明の抵抗性マーカーを含むことから、例えば、前記本発明の抵抗性マーカーの説明を援用できる。本発明において、前記第６染色体上のうどんこ病抵抗性遺伝子座は、例えば、本発明の抵抗性マーカーにおける抵抗性遺伝子座と読み替え可能である。本発明のうどんこ病抵抗性メロン植物は、例えば、前記本発明の抵抗性マーカー等の説明を援用できる。 The powdery mildew resistant melon plant of the present invention comprises the powdery mildew resistant locus on chromosome 6 in a homozygous form, and the powdery mildew resistant locus on chromosome 6 has the following conditions ( It is characterized in that at least one of the conditions 1) and (2) is satisfied, and other configurations and conditions are not particularly limited. Since the powdery mildew resistant melon plant of this invention contains the resistance marker of the said this invention which is the said resistance locus, description of the said resistance marker of this invention can be used, for example. In the present invention, the powdery mildew resistance locus on chromosome 6 can be read as the resistance locus in the resistance marker of the present invention, for example. For the powdery mildew resistant melon plant of the present invention, for example, the description of the resistance marker of the present invention can be incorporated.
　本発明のうどんこ病抵抗性メロン植物は、うどんこ病に抵抗性を示す。 The powdery mildew resistant melon plant of the present invention is resistant to powdery mildew.
　本発明のうどんこ病抵抗性メロン植物において、前記うどんこ病抵抗性は、前記抵抗性遺伝子座によってもたらされる。本発明のうどんこ病抵抗性メロン植物は、前記第６染色体上の抵抗性遺伝子座をホモ接合型で含むが、前記うどんこ病抵抗性メロン植物は、例えば、第６染色体に代えて、第６染色体以外のいずれの染色体上に、前記第６染色体上の抵抗性遺伝子座を含んでもよい。すなわち、前記うどんこ病抵抗性メロン植物は、第１染色体、第２染色体、第３染色体、第４染色体、第５染色体、第７染色体、第８染色体、第９染色体、第１０染色体、第１１染色体、第１２染色体のいずれかの染色体上に、前記第６染色体上の前記抵抗性遺伝子座を含んでもよい。前記うどんこ病抵抗性メロン植物が前記抵抗性マーカーを第６染色体以外の染色体上に含む場合、例えば、１つの抵抗性遺伝子座を第６染色体以外の染色体上に含んでもよいし、２つの抵抗性遺伝子座を第６染色体以外の染色体上に含んでもよい。後者の場合、前記うどんこ病抵抗性メロン植物は、例えば、前記２つの抵抗性遺伝子座を同じ染色体上に含んでもよいし、異なる染色体上に含んでもよい。なお、前記「染色体」は、例えば、前述のように、「連鎖群」ということもでき、前述の説明を援用できる。 In the powdery mildew resistant melon plant of the present invention, the powdery mildew resistance is brought about by the resistance locus. The powdery mildew resistant melon plant of the present invention includes the resistance locus on the sixth chromosome in a homozygous form, but the powdery mildew resistant melon plant includes, for example, a chromosome 6 instead of the sixth chromosome. The resistance locus on the sixth chromosome may be included on any chromosome other than the sixth chromosome. That is, the powdery mildew resistant melon plant has chromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5, chromosome 7, chromosome 8, chromosome 9, chromosome 10, The resistance locus on the sixth chromosome may be included on either the chromosome or the twelfth chromosome. When the powdery mildew resistant melon plant includes the resistance marker on a chromosome other than the sixth chromosome, for example, one resistance locus may be included on a chromosome other than the sixth chromosome, or two resistances may be included. Sex loci may be included on chromosomes other than chromosome 6. In the latter case, the powdery mildew resistant melon plant may include, for example, the two resistance loci on the same chromosome or different chromosomes. The “chromosome” can also be referred to as a “linkage group” as described above, for example, and the above description can be used.
　本発明のうどんこ病抵抗性メロン植物において、前記抵抗性遺伝子座は、例えば、前記本発明の抵抗性マーカーの説明を援用できる。 In the powdery mildew resistant melon plant of the present invention, the description of the resistance marker of the present invention can be cited as the resistance locus, for example.
　本発明のうどんこ病抵抗性メロン植物は、例えば、前記条件（１）および（２）の一方の条件を満たしてもよいし、両条件を満たしてもよく、うどんこ病抵抗性との相関性がより高いことから、両条件を満たすことが好ましい。 The powdery mildew resistant melon plant of the present invention may satisfy, for example, one of the conditions (1) and (2), or may satisfy both conditions, and correlation with powdery mildew resistance. It is preferable that both conditions are satisfied because the property is higher.
　本発明のうどんこ病抵抗性メロン植物は、一例として、受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物（Cucumis　melo）またはその後代系統があげられる。前記寄託メロン植物は、例えば、前記第６染色体上に前記抵抗性遺伝子座をホモ接合型で含む。寄託の情報を以下に示す。
寄託の種類：国際寄託
寄託機関名：独立行政法人製品評価技術基盤機構　特許生物寄託センター
あて名：日本国　〒２９２－０８１８　千葉県木更津市かずさ鎌足２－５－８　１２０号室
受託番号：ＦＥＲＭ　ＢＰ－２２２９１
識別のための表示：Ｔａｋｉｉ８
受領日：２０１５年８月１４日 An example of the powdery mildew resistant melon plant of the present invention is a melon plant ( Cucumis melo ) deposited under the accession number FERM BP-22291 or its progeny line. The deposited melon plant includes, for example, the resistance locus on the sixth chromosome in a homozygous form. The deposit information is shown below.
Deposit Type: International Depositary Agency Name: National Institute of Technology and Evaluation Patent Biological Depositary Center Address: Japan 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818 Room No. 120 Accession Number: FERM BP- 22291
Display for identification: Takii8
Date of receipt: August 14, 2015
　本発明のうどんこ病抵抗性メロン植物は、例えば、メロン植物に、前記抵抗性遺伝子座を導入することによっても製造できる。前記メロン植物への前記抵抗性遺伝子座の導入方法は、特に制限されず、例えば、従来公知の遺伝子工学的手法があげられる。導入する前記抵抗性遺伝子座は、例えば、前述のうどんこ病抵抗性遺伝子座が例示できる。 The powdery mildew resistant melon plant of the present invention can also be produced, for example, by introducing the resistance locus into a melon plant. The method for introducing the resistance locus into the melon plant is not particularly limited, and examples thereof include conventionally known genetic engineering techniques. Examples of the resistance locus to be introduced include the powdery mildew resistance locus described above.
　本発明のうどんこ病抵抗性メロン植物について、うどんこ病抵抗性以外の特徴、例えば、形質的、生態的特徴等は、特に限定されない。 Regarding the powdery mildew resistant melon plant of the present invention, characteristics other than powdery mildew resistance, such as trait and ecological characteristics are not particularly limited.
　本発明のうどんこ病抵抗性メロン植物は、さらに、他の抵抗性を有してもよい。 The powdery mildew resistant melon plant of the present invention may further have other resistance.
　本発明において、「植物体」は、植物全体を示す植物個体および前記植物個体の部分のいずれの意味であってもよい。前記植物個体の部分は、例えば、器官、組織、細胞または栄養繁殖体等があげられ、いずれでもよい。前記器官は、例えば、花弁、花冠、花、葉、種子、果実、茎、根等があげられる。前記組織は、例えば、前記器官の部分である。前記植物体の部分は、例えば、１種類の器官、組織および／または細胞でもよいし、２種類以上の器官、組織および／または細胞でもよい。 In the present invention, the “plant body” may mean either a plant individual indicating the whole plant or a part of the plant individual. Examples of the plant individual part include organs, tissues, cells, vegetative propagation bodies, and the like. Examples of the organ include petals, corolla, flowers, leaves, seeds, fruits, stems, roots and the like. The tissue is, for example, a part of the organ. The plant body part may be, for example, one kind of organ, tissue and / or cell, or two or more kinds of organ, tissue and / or cell.
３．うどんこ病抵抗性メロン植物の製造方法
　つぎに、本発明のうどんこ病抵抗性メロン植物の製造方法（以下、「製造方法」ともいう。）について説明する。なお、以下の方法は、例示であって、本発明は、これらの方法に制限されない。本発明において、製造方法は、例えば、育成方法ということもできる。また、本発明において、前記うどんこ病抵抗性遺伝子座は、前記本発明の抵抗性マーカーと言い換えることができる。 3. Next, a method for producing the powdery mildew resistant melon plant of the present invention (hereinafter also referred to as “manufacturing method”) will be described. The following methods are examples, and the present invention is not limited to these methods. In the present invention, the production method can also be referred to as a growth method, for example. In the present invention, the powdery mildew resistance locus can be rephrased as the resistance marker of the present invention.
　本発明のうどんこ病抵抗性メロン植物の製造方法は、前述のように、下記（ａ）および（ｂ）工程を含むことを特徴とする。
（ａ）前記本発明のうどんこ病抵抗性メロン植物と、他のメロン植物とを交雑する工程
（ｂ）前記（ａ）工程より得られたメロン植物またはその後代系統から、うどんこ病抵抗性メロン植物を選抜する工程 As described above, the method for producing a powdery mildew resistant melon plant of the present invention includes the following steps (a) and (b).
(A) Step of crossing the powdery mildew resistant melon plant of the present invention with another melon plant (b) Powdery mildew resistance from the melon plant obtained from the step (a) or its progeny line Process for selecting melon plants
　本発明の製造方法は、前記本発明のうどんこ病抵抗性メロン植物を親として使用することが特徴であって、その他の工程および条件は、特に制限されない。本発明の製造方法は、例えば、前記本発明の抵抗性マーカー等の説明を援用できる。前述のように、前記うどんこ病抵抗性遺伝子座は、例えば、単一の遺伝子座でうどんこ病抵抗性を付与できる。このため、本発明の製造方法は、例えば、前記抵抗性遺伝子座を用いることにより、他のメロン植物との交雑によって得られたＦ１またはその後代からも、うどんこ病抵抗性を示す後代を簡便に得ることができる。 The production method of the present invention is characterized by using the powdery mildew resistant melon plant of the present invention as a parent, and other steps and conditions are not particularly limited. For example, the description of the resistance marker of the present invention can be used in the production method of the present invention. As described above, the powdery mildew resistance locus can impart powdery mildew resistance at a single locus, for example. For this reason, the production method of the present invention, for example, by using the above-mentioned resistance locus, can easily produce progeny showing powdery mildew resistance from F1 obtained by crossing with other melon plants or from its progeny. Can get to.
　前記（ａ）工程において、第一の親として使用するうどんこ病抵抗性メロン植物は、前記本発明のうどんこ病抵抗性メロン植物であればよい。前記うどんこ病抵抗性メロン植物は、例えば、前述のような受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託されたメロン植物またはその後代系統が好ましい。前記（ａ）工程において、第一の親として使用するうどんこ病抵抗性メロン植物は、例えば、後述する本発明のスクリーニング方法により得ることもできる。このため、前記うどんこ病抵抗性メロン植物は、例えば、前記（ａ）工程に先立って、例えば、被検メロン植物（以下、「候補メロン植物」ともいう。）から、下記（ｘ）工程により選抜して準備してもよい。
（ｘ）被検メロン植物から、前記本発明のうどんこ病抵抗性メロン植物を選抜する工程 In the step (a), the powdery mildew resistant melon plant used as the first parent may be the powdery mildew resistant melon plant of the present invention. The powdery mildew resistant melon plant is preferably, for example, a melon plant deposited under the accession number FERM BP-22291 as described above or its progeny line. In the step (a), the powdery mildew resistant melon plant used as the first parent can be obtained, for example, by the screening method of the present invention described later. Therefore, the powdery mildew resistant melon plant is obtained, for example, from the test melon plant (hereinafter also referred to as “candidate melon plant”) by the following step (x) prior to the step (a). You may select and prepare.
(X) a step of selecting the powdery mildew resistant melon plant of the present invention from the test melon plant
　前記（ｘ）工程において、前記うどんこ病抵抗性メロン植物の選抜は、前記抵抗性遺伝子座を含むメロン植物の選抜ということができる。このため、前記（ｘ）工程は、例えば、下記（ｘ１）工程および（ｘ２）工程により行うことができる。
（ｘ１）前記被検メロン植物の染色体上における、ホモ接合型のうどんこ病抵抗性遺伝子座の有無を検出する検出工程
（ｘ２）前記ホモ接合型のうどんこ病抵抗性遺伝子座の存在により、前記被検メロン植物を、うどんこ病抵抗性メロン植物として選抜する選抜工程 In the step (x), selection of the powdery mildew resistant melon plant can be said to be selection of a melon plant containing the resistant locus. For this reason, the said (x) process can be performed by the following (x1) process and (x2) process, for example.
(X1) a detection step of detecting the presence or absence of a homozygous powdery mildew resistance locus on the chromosome of the test melon plant (x2) due to the presence of the homozygous powdery mildew resistance locus, Selection process for selecting the test melon plant as a powdery mildew resistant melon plant
　前記（ｘ）工程における前記選抜は、前述のように、例えば、前記うどんこ病抵抗性遺伝子座を含むメロン植物の選抜であり、具体的には、前記被検メロン植物について、前記うどんこ病抵抗性遺伝子座を検出することによって、前記うどんこ病抵抗性メロン植物を選抜できる。前記うどんこ病抵抗性遺伝子座の検出は、例えば、前記本発明の抵抗性マーカーにおいて説明したように、前記抵抗性遺伝子座が満たす、（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件、（ii）塩基配列に基づく条件、（iii）領域の塩基配列に基づく条件およびこれらの条件の組合せを用いて検出できる。 As described above, the selection in the step (x) is, for example, selection of a melon plant containing the powdery mildew resistance locus, and specifically, for the test melon plant, the powdery mildew By detecting the resistance locus, the powdery mildew resistant melon plant can be selected. The detection of the powdery mildew resistance locus is, for example, a condition based on a combination of the length of the amplified fragment and the SNP that the resistance locus satisfies, as described in the resistance marker of the present invention. , (Ii) conditions based on the base sequence, (iii) conditions based on the base sequence of the region, and combinations of these conditions.
　前記（ｘ）工程における前記選抜について、以下の具体例をあげるが、本発明は、これらには限定されない。また、前記うどんこ病抵抗性遺伝子座に関しては、前記本発明の抵抗性マーカーにおける説明を援用できる。 The following specific examples are given for the selection in the step (x), but the present invention is not limited to these. Moreover, regarding the powdery mildew resistance gene locus, the explanation for the resistance marker of the present invention can be used.
　前記（ｘ）工程における前記選抜は、例えば、第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含むうどんこ病抵抗性メロン植物の選抜であり、前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記条件（１）および（２）の少なくとも一方の条件を満たす。 The selection in the step (x) is, for example, selection of a powdery mildew resistant melon plant containing a powdery mildew resistant locus on chromosome 6 in a homozygous form, and powdery mildew on the sixth chromosome. The disease resistance locus satisfies at least one of the conditions (1) and (2).
　前記（ｘ）工程において、前記抵抗性遺伝子座は、前述のように、例えば、（ｉ）プライマーセットで増幅される増幅断片の長さおよびＳＮＰの組合せの少なくとも一方に基づく条件により選抜される。前記抵抗性遺伝子座は、例えば、さらに、後述するように、（ii）プライマーセットにより増幅される増幅断片の塩基配列およびＳＮＰの組合せを含む塩基配列の少なくとも一方に基づく条件により選抜されてもよいし、（iii）プライマーセットで増幅される増幅断片の塩基配列およびＳＮＰの組合せを含む塩基配列の少なくとも一方を含む領域の塩基配列に基づく条件により選抜されてもよいし、これらの組合せの条件により選抜されてもよい。前記組合せの条件により選抜する場合、前記組合せは、特に制限されず、例えば、以下の組合せが例示できる。また、前記抵抗性遺伝子座は、例えば、前記（ｉ）により選抜されるが、本発明はこれに限定されず、例えば、前記（ｉ）に代えて、前記（ii）または前記（iii）により選抜されてもよいし、前記（ii）および前記（iii）を組合せた条件により選抜されてもよい。
前記（ｉ）および前記（ii）の組合せ
前記（ｉ）および前記（iii）の組合せ
前記（ｉ）、前記（ii）、および前記（iii）の組合せ In the step (x), as described above, the resistance locus is selected based on, for example, (i) the condition based on at least one of the length of the amplified fragment amplified by the primer set and the combination of SNPs. The resistance locus may be selected, for example, under conditions based on at least one of the base sequence of the amplified fragment amplified by the primer set and the base sequence including the combination of SNPs, as will be described later. (Iii) may be selected based on conditions based on the base sequence of the amplified fragment amplified by the primer set and the base sequence of the region containing at least one of the base sequences including the combination of SNPs, or depending on the conditions of these combinations It may be selected. When selecting according to the conditions of the said combination, the said combination is not restrict | limited in particular, For example, the following combinations can be illustrated. The resistance locus is selected by, for example, (i). However, the present invention is not limited to this, and for example, instead of (i), by (ii) or (iii) It may be selected, or may be selected according to a combination of (ii) and (iii).
Combination of (i) and (ii) Combination of (i) and (iii) Combination of (i), (ii), and (iii)
（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件による選抜
　前記抵抗性遺伝子座において、前記選抜に用いる（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件は、特に制限されず、例えば、前記本発明の抵抗性マーカーにおける「（ｉ）増幅断片の長さおよびＳＮＰの組合せに基づく条件」の説明を援用できる。 (I) Selection based on conditions based on the combination of length of amplified fragment and SNP In the resistance locus, conditions based on the combination of length of amplified fragment and SNP used for the selection are not particularly limited, For example, the description of “(i) Conditions based on a combination of length of amplified fragment and SNP” in the resistance marker of the present invention can be cited.
　具体例として、前記（ｘ）工程における前記選抜は、例えば、前記条件（１）および（２）の一方の条件を満たすうどんこ病抵抗性メロン植物の選抜でもよいし、両条件を満たすうどんこ病抵抗性メロン植物の選抜でもよい。前記条件（２）において、前記抵抗性遺伝子座は、例えば、さらに、前記配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列（配列番号７：5’-AAAAGCTCCA-3’）により特定されてもよい。 As a specific example, the selection in the step (x) may be, for example, selection of a powdery mildew resistant melon plant that satisfies one of the conditions (1) and (2), or a powdery mildew that satisfies both conditions. It is also possible to select a disease-resistant melon plant. In the condition (2), the resistance locus is further specified by, for example, the 204th to 212th and 213th base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in the base sequence of SEQ ID NO: 3. May be.
　さらに、前記（ｘ）工程において、前記抵抗性遺伝子座は、例えば、前記ＳＮＰの組合せに基づく条件として、さらに、前記条件（３）および（４）の少なくとも一方の条件を満たしてもよい。また、前記条件（３）において、前記抵抗性遺伝子座は、例えば、さらに、前記配列番号４の塩基配列における１３２および４５７番目のＳＮＰの組合せにより特定されてもよい。 Furthermore, in the step (x), for example, the resistance locus may further satisfy at least one of the conditions (3) and (4) as a condition based on the combination of the SNPs. In the condition (3), the resistance locus may be further specified by, for example, a combination of the 132nd and 457th SNPs in the base sequence of SEQ ID NO: 4.
　前記抵抗性遺伝子座が、さらに、前記条件（３）および（４）の少なくとも一方の条件を満たす場合、前記抵抗性遺伝子座が満たす条件の組合せは、特に制限されず、例えば、前記本発明の抵抗性マーカーにおける組合せの例示を援用できる。 When the resistance locus further satisfies at least one of the conditions (3) and (4), the combination of the conditions satisfied by the resistance locus is not particularly limited. Examples of combinations in resistance markers can be cited.
　なお、本発明の製造方法において、前記抵抗性遺伝子座は、例えば、前記条件（１）および（２）の少なくとも一方の条件を満たすが、前記抵抗性遺伝子座はこれに限定されず、前記条件（１）および（２）の少なくとも一方の条件に代えて、前記条件（３）および（４）の少なくとも一方の条件を満たしてもよい。この場合、前記抵抗性遺伝子座は、例えば、前記条件（３）および（４）の一方の条件を満たしてもよいし、両条件を満たしてもよく、うどんこ病抵抗性との相関性がより高いことから、両条件を満たすことが好ましい。 In the production method of the present invention, the resistance locus satisfies, for example, at least one of the conditions (1) and (2), but the resistance locus is not limited thereto, and the conditions Instead of at least one of the conditions (1) and (2), at least one of the conditions (3) and (4) may be satisfied. In this case, for example, the resistance locus may satisfy one of the conditions (3) and (4), or may satisfy both conditions, and has a correlation with powdery mildew resistance. Since it is higher, it is preferable to satisfy both conditions.
（ii）塩基配列に基づく条件による選抜
　前記（ｘ）工程における選抜は、例えば、前記第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含むうどんこ病抵抗性メロン植物の選抜であり、前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記条件（５）および（６）の少なくとも一方の条件を満たしてもよい。前記抵抗性遺伝子座において、前記選抜に用いる（ii）塩基配列に基づく条件は、特に制限されず、例えば、前記本発明の抵抗性マーカーにおける「（ii）塩基配列に基づく条件」の説明を援用できる。 (Ii) Selection based on conditions based on nucleotide sequence Selection in the step (x) is, for example, selection of a powdery mildew resistant melon plant containing the powdery mildew resistant gene locus on chromosome 6 in a homozygous form. Yes, the powdery mildew resistance locus on the sixth chromosome may satisfy at least one of the conditions (5) and (6). In the resistance locus, conditions based on (ii) base sequence used for the selection are not particularly limited, and for example, the description of “(ii) conditions based on base sequence” in the resistance marker of the present invention is used. it can.
　具体例として、前記（ｘ）工程における前記選抜は、例えば、前記条件（５）および（６）の一方の条件を満たすうどんこ病抵抗性メロン植物の選抜でもよいし、両条件を満たすうどんこ病抵抗性メロン植物の選抜でもよい。 As a specific example, the selection in the step (x) may be, for example, selection of a powdery mildew-resistant melon plant that satisfies one of the conditions (5) and (6), or an noodle that satisfies both conditions It is also possible to select a disease-resistant melon plant.
　前記条件（６）において、前記（ｂ２）のポリヌクレオチドは、例えば、さらに、前記（ｂ１）における２０４～２１２および２１３番目の塩基配列（配列番号７：5’-AAAAGCTCCA-3’）が保存されてもよい。また、前記条件（６）において、前記（ｂ３）のポリヌクレオチドは、例えば、さらに、前記（ｂ１）における２０４～２１２および２１３番目の塩基配列（配列番号７：5’-AAAAGCTCCA-3’）が保存されてもよい。 In the condition (6), the polynucleotide (b2) further stores, for example, the 204th to 212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1). May be. In the condition (6), the polynucleotide (b3) further includes, for example, the 204-212th and 213rd base sequences (SEQ ID NO: 7: 5′-AAAAGCTCCA-3 ′) in (b1). It may be saved.
　さらに、前記（ｘ）工程において、前記抵抗性遺伝子座は、例えば、前記塩基配列に基づく条件として、さらに、前記条件（７）および（８）の少なくとも一方の条件を満たしてもよい。また、前記条件（７）において、前記（ｃ２）のポリヌクレオチドは、例えば、さらに、前記（ｃ１）における１３２番目の塩基（Ｔ）および４５７番目の塩基（Ａ）が保存されてもよい。また、前記条件（７）において、前記（ｃ３）のポリヌクレオチドは、例えば、さらに、前記（ｃ１）における１３２番目の塩基（Ｔ）および４５７番目の塩基（Ａ）が保存されてもよい。 Furthermore, in the step (x), the resistance locus may further satisfy at least one of the conditions (7) and (8) as a condition based on the base sequence, for example. In the condition (7), for example, in the polynucleotide (c2), the 132nd base (T) and the 457th base (A) in the (c1) may be further preserved. In the condition (7), the polynucleotide (c3) may further store, for example, the 132nd base (T) and the 457th base (A) in the (c1).
　前記抵抗性遺伝子座が、さらに、前記条件（７）および（８）の少なくとも一方の条件を満たす場合、前記抵抗性遺伝子座が満たす条件の組合せは、特に制限されず、例えば、前記本発明の抵抗性マーカーにおける組合せの例示を援用できる。 When the resistance locus further satisfies at least one of the conditions (7) and (8), the combination of the conditions satisfied by the resistance locus is not particularly limited. Examples of combinations in resistance markers can be cited.
（iii）領域の塩基配列に基づく条件による選抜
　前記（ｘ）工程における選抜は、例えば、前記第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含むうどんこ病抵抗性メロン植物の選抜であり、前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記条件（９）を満たしてもよい。前記抵抗性遺伝子座において、前記選抜に用いる（iii）領域の塩基配列に基づく条件は、特に制限されず、例えば、前記本発明の抵抗性マーカーにおける「（iii）領域の塩基配列に基づく条件」の説明を援用できる。 (Iii) Selection based on conditions based on the nucleotide sequence of the region The selection in the step (x) is, for example, that of a powdery mildew resistant melon plant containing the powdery mildew resistant locus on the sixth chromosome in a homozygous form. The powdery mildew resistance locus on the sixth chromosome may be selected, and the condition (9) may be satisfied. In the resistance gene locus, the conditions based on the nucleotide sequence of the (iii) region used for the selection are not particularly limited. For example, “conditions based on the nucleotide sequence of the (iii) region” in the resistance marker of the present invention. Can be used.
　具体例として、前記抵抗性遺伝子座は、前記領域の塩基配列において、例えば、前記条件（１）および（２）の少なくとも一方の条件を満たすことが好ましい。また、前記抵抗性遺伝子座は、さらに、前記領域の塩基配列において、例えば、前記条件（３）および（４）の少なくとも一方の条件を満たすことが好ましい。 As a specific example, the resistance locus preferably satisfies at least one of the conditions (1) and (2) in the base sequence of the region. Moreover, it is preferable that the resistance locus further satisfies, for example, at least one of the conditions (3) and (4) in the base sequence of the region.
　また、前記抵抗性遺伝子座は、例えば、前記条件（ａ）、（ｂ）または（ｃ）を満たしてもよい。 Further, the resistance locus may satisfy, for example, the condition (a), (b), or (c).
　前記ホモ接合型の抵抗性遺伝子座の有無を検出する染色体は、好ましくは、第６染色体である。 The chromosome for detecting the presence or absence of the homozygous resistance locus is preferably the sixth chromosome.
　また、前記（ａ）工程において、他方の親として使用するメロン植物は、特に制限されず、例えば、既知のうどんこ病抵抗性遺伝子を含むメロン植物でもよいし、他の抵抗性を有するメロン植物でもよいし、前記本発明のうどんこ病抵抗性メロン植物でもよい。 In the step (a), the melon plant used as the other parent is not particularly limited. For example, a melon plant containing a known powdery mildew resistance gene may be used, or a melon plant having other resistance. Alternatively, the powdery mildew resistant melon plant of the present invention may be used.
　前記（ａ）工程において、前記うどんこ病抵抗性メロン植物と前記他のメロン植物との交雑方法は、特に制限されず、公知の方法が採用できる。 In the step (a), the method of crossing the powdery mildew resistant melon plant with the other melon plant is not particularly limited, and a known method can be adopted.
　前記（ｂ）工程において、うどんこ病抵抗性メロン植物を選抜する対象は、例えば、前記（ａ）工程より得られたメロン植物でもよいし、さらに、そのメロン植物から得られた後代系統でもよい。具体的に、前記対象は、例えば、前記（ａ）工程の交雑によって得られたＦ１のメロン植物でもよいし、その後代系統でもよい。前記後代系統は、例えば、前記（ａ）工程の交雑によって得られたＦ１のメロン植物の自殖交雑後代または戻し交雑後代でもよいし、前記Ｆ１のメロン植物と他のメロン植物とを交雑することによって得られたメロン植物であってもよい。 In the step (b), the target for selecting the powdery mildew resistant melon plant may be, for example, a melon plant obtained from the step (a) or a progeny line obtained from the melon plant. . Specifically, the target may be, for example, an F1 melon plant obtained by crossing in the step (a) or a progeny line. The progeny line may be, for example, an inbred progeny or a backcross progeny of the F1 melon plant obtained by crossing in the step (a), or crossing the F1 melon plant with another melon plant. It may be a melon plant obtained by
　前記（ｂ）工程において、うどんこ病抵抗性メロン植物の選抜は、例えば、うどんこ病抵抗性を、直接的または間接的に確認することにより行うことができる。 In the step (b), selection of powdery mildew resistant melon plants can be performed, for example, by confirming powdery mildew resistance directly or indirectly.
　前記（ｂ）工程において、前記直接的な確認は、得られた前記Ｆ１のメロン植物またはその後代系統について、例えば、うどんこ病抵抗性を、前述のような発病指数によって評価することで行える。具体的には、例えば、前記Ｆ１のメロン植物またはその後代系統に対して、例えば、うどんこ病菌を接種して、うどんこ病抵抗性を、前記発病指数によって評価することで確認できる。この場合、例えば、１以下の発病度を示す前記Ｆ１のメロン植物またはその後代系統を、うどんこ病抵抗性メロン植物として選抜できる。 In the step (b), the direct confirmation can be performed by, for example, evaluating the powdery mildew resistance of the obtained F1 melon plant or its progeny line by the disease incidence index as described above. Specifically, for example, powdery mildew fungus is inoculated to the F1 melon plant or its progeny line, and powdery mildew resistance can be confirmed by evaluating the disease index. In this case, for example, the F1 melon plant having a disease severity of 1 or less or its progeny can be selected as a powdery mildew resistant melon plant.
　また、前記（ｂ）工程において、前記間接的な確認による選抜は、例えば、下記（ｂ１）および（ｂ２）工程によって行うことができる。
（ｂ１）前記（ａ）工程より得られたメロン植物またはその後代系統について、染色体上における、ホモ接合型のうどんこ病抵抗性遺伝子座の有無を検出する検出工程
（ｂ２）前記うどんこ病抵抗性遺伝子座の存在により、前記（ａ）工程により得られたメロン植物またはその後代系統を、うどんこ病抵抗性メロン植物として選抜する選抜工程 In the step (b), the selection by the indirect confirmation can be performed by, for example, the following steps (b1) and (b2).
(B1) Detection step for detecting presence or absence of homozygous powdery mildew resistance locus on the chromosome of the melon plant or its progeny obtained in the step (a) (b2) Powdery mildew resistance A selection step of selecting the melon plant obtained by the step (a) or its progeny line as a powdery mildew resistant melon plant due to the presence of the sex locus
　前記（ｂ）工程におけるうどんこ病抵抗性メロン植物の選抜は、例えば、前記（ｘ）工程において説明した方法と同様であり、前記ホモ接合型のうどんこ病抵抗性遺伝子座の有無の検出によって、より具体的には、前記分子マーカーを使用した前記ホモ接合型のうどんこ病抵抗性遺伝子座の有無の検出によって、行うことができる。 The selection of the powdery mildew resistant melon plant in the step (b) is, for example, the same as the method described in the step (x), by detecting the presence or absence of the homozygous powdery mildew resistant locus. More specifically, the detection can be performed by detecting the presence or absence of the homozygous powdery mildew resistance locus using the molecular marker.
　本発明の製造方法は、前記（ｂ）工程において選抜されたうどんこ病抵抗性メロン植物を、さらに育成することが好ましい。 In the production method of the present invention, it is preferable to further grow the powdery mildew resistant melon plant selected in the step (b).
　このように、前記うどんこ病抵抗性が確認された前記メロン植物またはその後代系統を、うどんこ病抵抗性メロン植物として選抜できる。 Thus, the melon plant or its progeny line confirmed to be resistant to powdery mildew can be selected as a powdery mildew resistant melon plant.
　本発明の製造方法は、さらに、交雑により得られた前記後代系統から、種子を採取する採種工程を含んでもよい。 The production method of the present invention may further include a seeding step of collecting seeds from the progeny line obtained by crossing.
４．うどんこ病抵抗性メロン植物のスクリーニング方法
　本発明のうどんこ病抵抗性メロン植物のスクリーニング方法（以下、「スクリーニング方法」ともいう。）は、交雑によりうどんこ病抵抗性メロン植物を生産するための親として、被検メロン植物から、メロン植物のうどんこ病抵抗性マーカーとして第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含むうどんこ病抵抗性メロン植物を選抜する工程を含み、
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする。
条件（１）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
前記増幅断片の長さが、１３３塩基長以上である。
プライマーセット１
　　　配列番号１の塩基配列からなるフォワードプライマー
　　　配列番号２の塩基配列からなるリバースプライマー
条件（２）
前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。 4). Screening Method for Powdery Mildew Resistant Melon Plant The screening method for powdery mildew resistant melon plant of the present invention (hereinafter also referred to as “screening method”) is for producing a powdery mildew resistant melon plant by crossing. As a parent, including a process for selecting a powdery mildew resistant melon plant containing a powdery mildew resistance locus on chromosome 6 in homozygous form as a powdery mildew resistance marker of a melon plant from a test melon plant ,
The powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
Condition (1)
The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
The length of the amplified fragment is 133 bases or more.
Primer set 1
Forward primer consisting of the base sequence of SEQ ID NO: 1 Reverse primer condition consisting of the base sequence of SEQ ID NO: 2 (2)
The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
　本発明のスクリーニング方法は、被検メロン植物から、メロン植物のうどんこ病抵抗性マーカーとして第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含むうどんこ病抵抗性メロン植物を選抜する工程を含み、前記第６染色体上のうどんこ病抵抗性遺伝子座が、前記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とし、その他の工程および条件は、特に制限されない。本発明のスクリーニング方法によれば、前記本発明の抵抗性マーカーによって、うどんこ病抵抗性の親を得ることができる。本発明のスクリーニング方法は、例えば、前記本発明の抵抗性マーカー等の説明を援用できる。 In the screening method of the present invention, a powdery mildew resistant melon plant containing a powdery mildew resistance locus on chromosome 6 in a homozygous form as a powdery mildew resistance marker of a melon plant is selected from test melon plants. Wherein the powdery mildew resistance locus on chromosome 6 satisfies at least one of the conditions (1) and (2), and the other steps and conditions are particularly limited. Not. According to the screening method of the present invention, a powdery mildew resistant parent can be obtained by the resistance marker of the present invention. For the screening method of the present invention, for example, the description of the resistance marker of the present invention can be used.
　前記親の選抜は、例えば、前記本発明のうどんこ病抵抗性メロン植物の製造方法における前記（ｘ）工程の説明を援用できる。 For the selection of the parent, for example, the description of the step (x) in the method for producing a powdery mildew resistant melon plant of the present invention can be used.
　以下、実施例を用いて本発明を詳細に説明するが、本発明は実施例に記載された態様に限定されるものではない。 Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to the modes described in the examples.
［実施例１］
　新規なうどんこ病抵抗性メロン植物について、うどんこ病抵抗性遺伝子座の遺伝様式の解析、前記うどんこ病抵抗性遺伝子座の特定、および前記うどんこ病抵抗性遺伝子座とうどんこ病抵抗性との相関を確認することで、前記うどんこ病抵抗性遺伝子座が、メロン植物のうどんこ病抵抗性マーカーとなること、前記抵抗性遺伝子座を含むメロン植物が、うどんこ病抵抗性メロン植物であること、ならびに前記メロン植物のうどんこ病抵抗性マーカーを使用し、うどんこ病抵抗性メロン植物をスクリーニングできることを確認した。 [Example 1]
Analysis of the inheritance pattern of the powdery mildew resistance locus, identification of the powdery mildew resistance locus, and the powdery mildew resistance locus and powdery mildew resistance of the novel powdery mildew resistant melon plant By confirming the correlation with the powdery mildew disease locus, the powdery mildew resistance locus is a powdery mildew resistance marker for melon plants, and the melon plant containing the resistance locus is a powdery mildew resistant melon plant. It was confirmed that the powdery mildew resistant melon plant can be screened using the powdery mildew resistance marker of the melon plant.
（１）寄託系統
　うどんこ病抵抗性を示す新規メロン植物を開発するために、タキイ種苗株式会社農場で継代育種により採取された大量のメロン系統の種子について、育種を行い、うどんこ病抵抗性の試験を行った。その結果、うどんこ病抵抗性を示す、新規のうどんこ病抵抗性メロン系統（Cucumis　melo）を製造（以下、「生産」ともいう。）した。この新規うどんこ病抵抗性メロン植物は、受託番号ＦＥＲＭ　ＢＰ－２２２９１で寄託した。以下、このうどんこ病抵抗性メロン植物を寄託系統という。 (1) Deposited line In order to develop a new melon plant exhibiting resistance to powdery mildew, seeds of a large number of melon lines collected by subculturing at Takii Seed Co., Ltd. farm are bred for powdery mildew resistance. A sex test was performed. As a result, a new powdery mildew resistant melon line ( Cucumis melo ) showing powdery mildew resistance was produced (hereinafter also referred to as “production”). This new powdery mildew resistant melon plant was deposited under accession number FERM BP-22291. Hereinafter, the powdery mildew resistant melon plant is referred to as a deposited line.
（２）うどんこ病抵抗性の遺伝様式
　前記寄託系統のメロン植物（受託番号ＦＥＲＭ　ＢＰ－２２２９１）と、うどんこ病罹病性メロン植物「春系３号（農業生物資源ジ－ンバンクからアクセッション番号ＪＰ３２０９７の植物として入手可能、Cucumis　melo）」（以下、「罹病性メロン植物」ともいう。）とを交雑することによって、６１個体のＦ２分離集団（以下、「６１系統」ともいう。）を生産した。さらに、前記６１系統を使用し、以下に示すように、うどんこ病菌の接種試験を行った。なお、前記寄託系統は、第６染色体上の前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７、前記ＳＮＰ２５０、前記ＳＮＰ２１３２、前記ＳＮＰ２１４１、前記ＳＮＰ２２６６、および前記ＳＮＰ２４５７、ならびに前記ＳＮＰ３９９、前記ＳＮＰ３１７４、前記ＳＮＰ３１８４、前記ＳＮＰ３１９９、および前記ＳＮＰ３２００を抵抗性のホモ接合型で含む。すなわち、前記寄託系統は、前記条件（１）～（８）および（９）を満たすメロン植物である。また、前記寄託系統は、２本の第６染色体が前記配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列を含み、且つ、前記プライマーセットで増幅される増幅断片の長さが、１３３塩基である。他方、前記罹病性メロン植物は、第６染色体上の前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７、前記ＳＮＰ２５０、前記ＳＮＰ２１３２、前記ＳＮＰ２１４１、前記ＳＮＰ２２６６、および前記ＳＮＰ２４５７、ならびに前記ＳＮＰ３９９、前記ＳＮＰ３１７４、前記ＳＮＰ３１８４、前記ＳＮＰ３１９９、および前記ＳＮＰ３２００を罹病性のホモ接合型で含む。また、前記罹病性メロン植物は、２本の第６染色体が前記配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列を含まず、且つ、前記プライマーセットで増幅される増幅断片の長さが、１３２塩基である。すなわち、前記罹病性メロン植物は、前記条件（１）～（８）および（９）を満たさないメロン植物である。 (2) Mode of inheritance of powdery mildew resistance The melon plant (Accession No. FERM BP-22291) of the deposited strain and the melon plant susceptible to powdery mildew “Spring 3 (accession number from Genebank, Agricultural Biological Resources Genebank) JP 32097 plant, Cucumis melo ) ”(hereinafter also referred to as“ susceptible melon plant ”) is crossed to produce 61 F2 segregated populations (hereinafter also referred to as“ 61 strains ”). did. Further, the 61 strains were used and an inoculation test for powdery mildew was performed as shown below. The deposited lines are the SNP1 45 , SNP1 48 , SNP1 49 , SNP1 51 , SNP1 108 , SNP1 120 , SNP1 139 , SNP1 214 , SNP1 327 , SNP2 50 , SNP2 132 , SNP2 141 , SNP2 266 , and SNP2 457 , and SNP3 99 , SNP3 174 , SNP3 184 , SNP3 199 , and SNP3 200 in a resistive homojunction type . That is, the deposited line is a melon plant that satisfies the conditions (1) to (8) and (9). In the deposited line, two chromosomes 6 include the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3, and the length of the amplified fragment amplified by the primer set is as follows: 133 bases. On the other hand, the susceptible melon plant is the SNP1 45 , SNP1 48 , SNP1 49 , SNP1 51 , SNP1 108 , SNP1 120 , SNP1 139 , SNP1 214 , and SNP1 327 on chromosome 6. The SNP2 50 , the SNP2 132 , the SNP2 141 , the SNP2 266 , and the SNP2 457 , and the SNP3 99 , the SNP3 174 , the SNP3 184 , the SNP3 199 , and the SNP3 200 Including. Further, the susceptible melon plant has two chromosomes 6 that do not contain the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3, and the length of the amplified fragment amplified by the primer set. Is 132 bases. That is, the susceptible melon plant is a melon plant that does not satisfy the conditions (1) to (8) and (9).
　うどんこ病菌の接種試験は、前記参考文献２に記載の方法を参照し、以下のように行った。 The inoculation test for powdery mildew was performed as follows with reference to the method described in Reference 2.
　前記うどんこ病菌系統１（レース３．５）は、隔離した培養器内で生育させたメロン植物（オトメの祈り、タキイ種苗株式会社）上で培養した。前記メロン植物の葉上の前記うどんこ病菌の菌叢を掻き取ることにより分生子を回収後、滅菌蒸留水を添加した。前記添加後、得られた懸濁液をキムワイプ（登録商標）で濾過し、濾液を回収した。さらに、前記分生子が、５×１０４個／ｍＬとなるように滅菌蒸留水で希釈し、分生子懸濁液を調製し、接種源として使用した。前記６１系統は、予めもみ殻燻炭に播種後、殺菌土壌を詰めた１００００分の１ａワグネルポットに鉢植えした。そして、前記接種試験には、本葉第３展開期のメロン植物を使用した。ハンドスプレーを用い、前記分生子懸濁液を前記メロン植物の株全体に均一に噴霧した。前記噴霧後、前記メロン植物を、２０－２５℃、湿度６０－８０％、１４０００Ｌｕｘ、１２時間日長の条件下の人工気象室において、２週間生育した。そして、生育したメロン植物について、以下のように発病調査を行った。また、１０系統の寄託系統および１０系統の前記罹病性メロン植物について、同様に発病調査を行った。なお、前記うどんこ病菌のレースは、下記参考文献３に基づき、決定した。
参考文献３：Fernando　J.　Yuste-Lisbona　et.al.,　“Codominant　PCR-based　markers　and　candidate　genes　for　powdery　mildew　resistance　in　melon　(Cucumis　melo　L.)”,　Theor.　Appl.　Genet.,　2011,　vol.122,　pp.747-758 The powdery mildew strain 1 (Race 3.5) was cultured on a melon plant (Otome no Prayer, Takii Seed Co., Ltd.) grown in an isolated incubator. After collecting the conidia by scraping the flora of the powdery mildew fungus on the leaves of the melon plant, sterile distilled water was added. After the addition, the resulting suspension was filtered through Kimwipe (registered trademark), and the filtrate was recovered. Further, the conidia were diluted with sterilized distilled water so as to be 5 × 10 4 cells / mL to prepare a conidia suspension, which was used as an inoculation source. The 61 strains were previously planted in rice husk charcoal and then potted in 1 / 10,000 Wagner pots filled with sterilized soil. In the inoculation test, a melon plant in the third development stage of the main leaf was used. Using a hand spray, the conidial suspension was sprayed uniformly over the entire strain of melon plants. After the spraying, the melon plant was grown for 2 weeks in an artificial weather room under conditions of 20-25 ° C., humidity 60-80%, 14000 Lux, 12 hours of day length. And about the grown melon plant, the disease investigation was conducted as follows. In addition, the pathogenesis of the 10 deposited lines and the 10 susceptible melon plants were similarly investigated. The race of the powdery mildew fungus was determined based on the following Reference 3.
Reference 3: Fernando J. Yuste-Lisbona et.al., “Codominant PCR-based markers and candidate genes for powdery mildew resistance in melon (Cucumis melo L.)”, Theor. Appl. Genet., 2011, vol.122 , pp.747-758
　発病調査は、前記参考文献２の方法を参照し、メロン植物の調査した葉の発病指数を、以下の基準にしたがって評価した。また、図２に、前記発病指数の評価基準として、発病指数０（図２（Ａ））、発病指数１（図２（Ｂ））、発病指数２（図２（Ｃ））、および発病指数３（図２（Ｄ））のメロン植物の葉の代表例の写真に示す。なお、各図において、胞子形成が観察される領域は、矢印で示した囲んだ領域である。
発病指数０：胞子形成が認められない（高い抵抗性）
発病指数１：わずかな胞子形成が認められる（抵抗性）
発病指数２：限定的な胞子形成が見られる
発病指数３：広範囲に多量の胞子形成が見られる For the disease investigation, the method of Reference 2 was referred to, and the disease incidence index of the leaves investigated by the melon plant was evaluated according to the following criteria. FIG. 2 shows the evaluation index of the disease index as the disease index 0 (FIG. 2A), disease index 1 (FIG. 2B), disease index 2 (FIG. 2C), and disease index. 3 (FIG. 2 (D)) shows a photograph of a representative example of leaves of a melon plant. In each figure, the area where spore formation is observed is the enclosed area indicated by the arrow.
Disease index 0: No sporulation observed (high resistance)
Disease index 1: Slight sporulation is observed (resistance)
Onset index 2: Onset of limited sporulation Onset index 3: Extensive sporulation over a wide range
　これらの結果を下記表１に示す。下記表１に示すように、前記６１系統において、発病指数が１以下、すなわちうどんこ病抵抗性である個体は、１５個体であり、全体の２４．６％を占めた。前記６１系統の発病指数と、その個体の出現頻度との関係から、前記寄託系統のうどんこ病抵抗性の遺伝様式は、１因子の劣性であることがわかった。また、同条件で試験した前記罹病性メロン植物は、罹病性を示した。前記寄託系統は、抵抗性を示すことが確認できた。さらに、１因子の劣性のうどんこ病抵抗性遺伝子座は知られていないことから、前記寄託系統が含むうどんこ病抵抗性遺伝子座は、新規なうどんこ病抵抗性遺伝子座であることが分かった。 These results are shown in Table 1 below. As shown in Table 1 below, in the 61 lines, the disease index was 1 or less, that is, the powdery mildew resistant individual was 15 individuals, accounting for 24.6% of the total. From the relationship between the disease index of the 61 strains and the frequency of appearance of the individuals, it was found that the inheritance pattern of powdery mildew resistance of the deposited strain was one factor recessive. Moreover, the susceptible melon plant tested under the same conditions showed susceptibility. It was confirmed that the deposited line showed resistance. Furthermore, since one factor of recessive powdery mildew resistance loci is not known, it was found that the powdery mildew resistance loci contained in the deposited strain are novel powdery mildew resistance loci. It was.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（３）新規なうどんこ病抵抗性遺伝子座の特定
　つぎに、前記６１系統から、常法により、それぞれの系統のＤＮＡを抽出した。さらに、前記ＤＮＡについて、ＤＮＡアッセイに供した。そして、前記（２）の発病指数および前記ＤＮＡアッセイの結果に基づき、解析ソフトウェア（QTL　cartographer、NC　STATE　University製）を用い、ＱＴＬ解析を行った。その結果、第６染色体において、前記発病指数と相関性の高い領域が、１箇所特定された。前記相関性の高い領域は、前記プライマーセット１で増幅される増幅断片および第１ＳＮＰセットを含む領域であった。これらの結果から、新規なうどんこ病抵抗性遺伝子座は、第６染色体上の１因子の劣性遺伝子を含むことが分かった。また、前記相関性の高い領域の塩基配列の解析から、うどんこ病抵抗性メロン植物は、２本の第６染色体から前記プライマーセット１で増幅される増幅断片の長さが１３３塩基であり、前記２本の第６染色体上において、前記ＳＮＰ１４５、前記ＳＮＰ１４８、前記ＳＮＰ１４９、前記ＳＮＰ１５１、前記ＳＮＰ１１０８、前記ＳＮＰ１１２０、前記ＳＮＰ１１３９、前記ＳＮＰ１２１４、および前記ＳＮＰ１３２７が、それぞれ、Ａ、Ａ、Ｔ、Ｔ、Ｔ、Ａ、Ａ、ＴおよびＣであり、前記２本の第６染色体が、それぞれ、前記配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列を含むことがわかった。 (3) Identification of novel powdery mildew resistance loci Next, DNA of each strain was extracted from the 61 strains by a conventional method. Further, the DNA was subjected to a DNA assay. Then, based on the disease index of (2) and the results of the DNA assay, QTL analysis was performed using analysis software (QTL cartographer, manufactured by NC STATE University). As a result, in the sixth chromosome, one region having a high correlation with the disease index was identified. The highly correlated region was a region containing the amplified fragment amplified with the primer set 1 and the first SNP set. From these results, it was found that the novel powdery mildew resistance locus contains a one-factor recessive gene on chromosome 6. Further, from the analysis of the base sequence of the highly correlated region, the powdery mildew resistant melon plant has a length of 133 bases of the amplified fragment amplified from two chromosomes 6 by the primer set 1, On the two chromosomes 6, the SNP1 45 , the SNP1 48 , the SNP1 49 , the SNP1 51 , the SNP1 108 , the SNP1 120 , the SNP1 139 , the SNP1 214 , and the SNP1 327 are respectively A, A, T, T, T, A, A, T, and C, and the two chromosomes 6 include the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3, respectively. I understood it.
（４）新規なうどんこ病抵抗性遺伝子座とうどんこ病抵抗性
　前記６１系統のＤＮＡについて、前記プライマーセット１を用いＰＣＲを行い、増幅された増幅断片の長さを測定した。また、前記６１系統のＤＮＡについて、下記プライマーセット２を用いＰＣＲを行い、増幅された増幅断片において、前記第１ＳＮＰセットに対応する多型の塩基を特定した。なお、前記プライマーセット１を用いた増幅は、９４℃で３分間インキュベート後、９４℃３０秒、５７℃３０秒、および７２℃６０秒を１サイクルとし、３５サイクル行い、前記３５サイクル後、７２℃で３分間インキュベートすることにより実施した。また、前記プライマーセット２を用いた増幅は、９４℃で３分間インキュベート後、９４℃３０秒、５６℃３０秒、および７２℃６０秒を１サイクルとし、３５サイクル行い、前記３５サイクル後、７２℃で３分間インキュベートすることにより実施した。そして、前記増幅断片の長さおよび前記第１ＳＮＰセットに対応する多型の塩基により、前記６１系統を抵抗性のホモ接合型（Ａ）、ヘテロ接合型（Ｈ）、および罹病性のホモ接合型（Ｂ）に分類した。これらの結果を下記表２に示す。なお、下記表２において、Ａは、２本の第６染色体から増幅される前記増幅断片の長さが共に１３３塩基以上であり、且つ前記第１ＳＮＰセットを抵抗性のホモ接合型で有する個体を示す。また、Ｈは、１本の第６染色体から増幅される増幅断片の長さが１３３塩基以上であり、他方の第６染色体から増幅される増幅断片の長さが１３３塩基未満であり、且つ、前記第１ＳＮＰセットをヘテロ接合型で有する個体を示す。さらに、Ｂは、２本の第６染色体から増幅される前記増幅断片の長さが共に１３３塩基未満であり、且つ前記第１ＳＮＰセットを罹病性のホモ接合型で有する個体を示す。 (4) Novel Powdery Mildew Resistance Locus and Powdery Mildew Resistance The 61 lines of DNA were subjected to PCR using the primer set 1, and the length of the amplified fragment was measured. Further, PCR was performed on the 61 strains of DNA using the primer set 2 below, and polymorphic bases corresponding to the first SNP set were identified in the amplified fragments. The amplification using the primer set 1 was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 57 ° C. for 30 seconds, and 72 ° C. for 60 seconds. This was carried out by incubating at 3 ° C. for 3 minutes. The amplification using the primer set 2 was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 60 seconds. After the 35 cycles, This was carried out by incubating at 3 ° C. for 3 minutes. Based on the length of the amplified fragment and the polymorphic base corresponding to the first SNP set, the 61 strains are resistant homozygous (A), heterozygous (H), and susceptible homozygous. Classified into (B). These results are shown in Table 2 below. In Table 2 below, A represents an individual in which the lengths of the amplified fragments amplified from two chromosomes 6 are 133 bases or more and the first SNP set is a resistant homozygous type. Show. In addition, the length of the amplified fragment amplified from one chromosome 6 is 133 bases or more, and the length of the amplified fragment amplified from the other chromosome 6 is less than 133 bases, and H 3 shows an individual having the first SNP set in a heterozygous form. Furthermore, B shows the individual | organism | solid which the length of the said amplified fragment amplified from two 6th chromosomes is less than 133 bases, and has the said 1st SNP set in a diseased homozygote type | mold.
プライマーセット２
　　　フォワードプライマー２　5’-GGAAAAATGCAGGGGAAG-3’　　（配列番号８）
　　　リバースプライマー２　　5’-CTGCCAAAAGCGACTTAACC-3’　（配列番号９） Primer set 2
Forward primer 2 5'-GGAAAAATGCAGGGGAAG-3 '(SEQ ID NO: 8)
Reverse primer 2 5'-CTGCCAAAAGCGACTTAACC-3 '(SEQ ID NO: 9)
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　前記表２に示すように、Ａでは、全ての個体が、発病指数が１以下であった。他方、ＨおよびＢは、全ての個体が、発病指数が２以上であった。これらの結果から、前記増幅断片の長さおよび前記第１ＳＮＰセットで特定される新規なうどんこ病抵抗性遺伝子座が、うどんこ病抵抗性を担っていることが確認できた。また、前記増幅断片の長さおよび前記第１ＳＮＰセットで特定される抵抗性遺伝子座、すなわち、前記条件（１）および（２）の両条件を満たす抵抗性遺伝子座が、うどんこ病抵抗性を担っていることから、前記抵抗性遺伝子座が、メロン植物のうどんこ病抵抗性マーカーとなること、ならびに前記メロン植物のうどんこ病抵抗性マーカーを使用し、うどんこ病抵抗性メロン植物をスクリーニングできることがわかった。 As shown in Table 2 above, in A, all individuals had a disease index of 1 or less. On the other hand, for H and B, all individuals had a disease index of 2 or more. From these results, it was confirmed that the length of the amplified fragment and the novel powdery mildew resistance locus specified by the first SNP set are responsible for powdery mildew resistance. Further, the resistance locus specified by the length of the amplified fragment and the first SNP set, that is, the resistance locus satisfying both of the conditions (1) and (2) has resistance to powdery mildew. Therefore, the resistance locus serves as a powdery mildew resistance marker for melon plants, and powdery mildew resistant melon plants are screened using the powdery mildew resistance marker for melon plants. I knew it was possible.
（５）公知のうどんこ病菌への抵抗性の確認
　前記６１系統から、前記抵抗性のホモ接合型の個体１０系統を選抜した。つぎに、前記１０系統をそれぞれ自殖させることにより、Ｆ３選抜系統を生産した。さらに、１０系統のＦ３選抜系統（Ｆ３－１～Ｆ３－１０）について、それぞれ１０個体使用し、前記（２）と同様にして、うどんこ病菌の接種試験を行った。そして、Ｆ３選抜系統の各系統について発病指数を求め、下記式より、それぞれ対応するＦ３選抜系統の発病指数とした。 (5) Confirmation of resistance to known powdery mildew fungus From the 61 lines, 10 lines of the resistant homozygous individuals were selected. Next, the F3 selection line was produced by self-breeding the 10 lines. Further, 10 individual F3 selected lines (F3-1 to F3-10) were used, and an inoculation test for powdery mildew was performed in the same manner as in (2) above. And the disease index was calculated | required about each system | strain of F3 selection system | strain, and it was set as the disease index of each corresponding F3 selection system | strain from the following formula.
発病指数＝［（０×ｎ０）＋（１×ｎ１）＋（２×ｎ２）＋（３×ｎ３）］／調査個体数
前記式において、「０、１、２、３」は、それぞれ発病指数を示し、「ｎ０、ｎ１、ｎ２、ｎ３」は、それぞれ、発病指数０、発病指数１、発病指数２、発病指数３の個体数を示す。 Disease index = [(0 × n 0 ) + (1 × n 1 ) + (2 × n 2 ) + (3 × n 3 )] / number of surveyed individuals In the above formula, “0, 1, 2, 3” is Respectively, “n 0 , n 1 , n 2 , n 3 ” indicates the number of individuals of the disease index 0, the disease index 1, the disease index 2, and the disease index 3, respectively.
　また、前記うどんこ病菌の接種試験では、前記うどんこ病菌系統１に加え、うどんこ病菌系統２（レース２Ｆ）、うどんこ病菌系統３（レース５）、うどんこ病菌系統４（レースＧ）をそれぞれ使用して実施した。また、前記Ｆ３選抜系統に代えて、公知のうどんこ病罹病性メロン植物であるVedrantais（Ｗｅｂサイト（http://www.burpee.com/heirloom-seeds-and-plants/heirloom-melons/melon-vedrantais-prod002040.html）において入手可能）、PMR45（PI601383としてUSDA（United　States　Department　of　Agriculture）から入手可能）、および前記罹病性メロン植物を、公知のうどんこ病抵抗性であるPMR5（Ames26809としてUSDAから入手可能）、WMR29（INRA（Institut　National　de　la　Recherche　Agronomique）から入手可能）、Edisto47（NSL34600としてUSDAから入手可能）、PI414723（USDAから入手可能）、MR1（INRAから入手可能）、およびPI124112（USDAから入手可能）、ならびに前記寄託系統を用いた以外は同様にしてうどんこ病菌の接種試験を行い、発病指数を評価した。なお、前記うどんこ病菌のレースは、前記参考文献３に基づき、決定した。これらの結果を表３に示す。 In addition, in the inoculation test for the powdery mildew fungus, in addition to the powdery mildew strain 1, the powdery mildew strain 2 (race 2F), the powdery mildew strain 3 (race 5), and the powdery mildew strain 4 (race G) Each was performed using. Further, instead of the F3 selection line, Vedrantais (Website (http://www.burpee.com/heirloom-seeds-and-plants/heirloom-melons/melon-), which is a known powdery mildew-susceptible melon plant. available from vedrantais-prod002040.html), PMR45 (available from USDA (United States Department of Agriculture) as PI601383), and the susceptible melon plant, PMR5 (Ames26809 as Ames26809), which is a known powdery mildew resistance ), WMR29 (available from INRA (available from Institut National de la Recherche Agronomique)), Edisto47 (available from USDA as NSL34600), PI414723 (available from USDA), MR1 (available from INRA), and PI124112 ( (Available from USDA), and the inoculation test of powdery mildew fungus was conducted in the same manner except that the deposited strain was used, and the disease index was evaluated. The race for the powdery mildew was determined based on the reference 3. These results are shown in Table 3.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　前記表３に示すように、公知のうどんこ病罹病性メロン植物であるVedrantais、PMR45、および前記罹病性メロン植物は、いずれのうどんこ病菌に対しても罹病性を示した。また、公知のうどんこ病抵抗性メロン植物であるPMR5およびPI124112は、前記うどんこ病菌系統２および３に対して抵抗性を示したのに対し、前記うどんこ病菌系統１および４に対しては罹病性を示した。WMR29およびEdisto47は、前記うどんこ病菌系統２に対して抵抗性を示したのに対し、前記うどんこ病菌系統１、３、および４に対しては罹病性を示した。PI1414723は、前記うどんこ病菌系統１～３に対して抵抗性を示したのに対し、前記うどんこ病菌系統４に対して罹病性を示した。MR1は、前記うどんこ病菌系統２～４に対して抵抗性を示したのに対し、前記うどんこ病菌系統１に対しては罹病性を示した。これに対し、前記Ｆ３選抜系統および前記寄託系統は、いずれのうどんこ病菌に対しても、抵抗性を示した。さらに、前記うどんこ病菌系統１～４は、いずれも公知のうどんこ病抵抗性遺伝子を含むメロン植物に対して感染可能なうどんこ病菌であることから、前記抵抗性遺伝子座は、公知のうどんこ病抵抗性遺伝子を含むメロン植物に対して感染可能なうどんこ病菌に対しても有効であることがわかった。これらの結果から、前記Ｆ３選抜系統および前記寄託系統が、公知の複数のレースのうどんこ病菌に対して抵抗性を示すことがわかった。また、公知のうどんこ病抵抗性メロン植物は、うどんこ病菌系統１～４のいずれかに対して罹病性を示すのに対し、前記Ｆ３選抜系統および前記寄託系統は、うどんこ病菌系統１～４のいずれに対しても抵抗性を示した。これらの結果から、前記条件（１）および（２）の両条件を満たす抵抗性遺伝子座が、公知のうどんこ病抵抗性メロン植物が含む抵抗性遺伝子座と異なる新規のうどんこ病抵抗性遺伝子座であることが確認できた。 As shown in Table 3, Vedrantais and PMR45, which are known powdery mildew susceptible melon plants, and the susceptible melon plant showed susceptibility to any powdery mildew fungus. The known powdery mildew resistant melon plants PMR5 and PI124112 showed resistance to the powdery mildew strains 2 and 3, whereas the powdery mildew strains 1 and 4 were resistant to the powdery mildew strain 1 and 4. Showed susceptibility. WMR29 and Edisto47 were resistant to the powdery mildew strain 2 whereas the powdery mildew strains 1, 3, and 4 were susceptible. PI1414723 was resistant to the powdery mildew strain 1 to 3, whereas it was susceptible to the powdery mildew strain 4. MR1 showed resistance to the powdery mildew strains 2 to 4, whereas it showed susceptibility to the powdery mildew strain 1. On the other hand, the F3 selection line and the deposited line showed resistance to any powdery mildew. Furthermore, since the powdery mildew strains 1 to 4 are all powdery mildews capable of infecting melon plants containing a known powdery mildew resistance gene, the resistance locus is a known powdery mildew. It was also found effective against powdery mildew fungus capable of infecting melon plants containing the disease resistance gene. From these results, it was found that the F3 selection line and the deposited line are resistant to a plurality of known powdery mildew fungi of races. In addition, known powdery mildew resistant melon plants show susceptibility to any of powdery mildew strains 1 to 4, whereas the F3 selected strain and the deposited strain are powdery mildew strains 1 to 4. Resistance to any of 4 was shown. From these results, a novel powdery mildew resistance gene in which the resistance locus satisfying both conditions (1) and (2) is different from the resistance locus contained in the known powdery mildew resistant melon plant It was confirmed that it was a seat.
（６）第６染色体上のうどんこ病抵抗性遺伝子座
　前記（２）と同様にして、Ｆ２分離集団を作製した。そして、前記Ｆ２分離集団について、前記（４）と同様にして、前記増幅断片の長さおよび前記第１ＳＮＰセットに対応する多型の塩基を特定した。さらに、前記Ｆ２分離集団のＤＮＡについて、下記プライマーセット３および４を用い、ＰＣＲを行い、増幅された増幅断片において、前記第２ＳＮＰセットおよび前記第３ＳＮＰセットに対応する多型の塩基を特定した。なお、前記プライマーセット３を用いた増幅は、９４℃で３分間インキュベート後、９４℃３０秒、５１℃３０秒、および７２℃６０秒を１サイクルとし、３５サイクル行い、前記３５サイクル後、７２℃で３分間インキュベートすることにより実施した。また、前記プライマーセット４を用いた増幅は、９４℃で３分間インキュベート後、９４℃３０秒、５８℃３０秒、および７２℃６０秒を１サイクルとし、３５サイクル行い、前記３５サイクル後、７２℃で３分間インキュベートすることにより実施した。 (6) Powdery mildew resistance locus on chromosome 6 F2 segregated population was prepared in the same manner as (2) above. For the F2 segregated population, the length of the amplified fragment and the polymorphic base corresponding to the first SNP set were identified in the same manner as in (4). Furthermore, PCR was performed on the DNA of the F2 isolated population using the following primer sets 3 and 4, and polymorphic bases corresponding to the second SNP set and the third SNP set were identified in the amplified fragments. The amplification using the primer set 3 was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 51 ° C. for 30 seconds, and 72 ° C. for 60 seconds. This was carried out by incubating at 3 ° C. for 3 minutes. The amplification using the primer set 4 was carried out at 94 ° C. for 3 minutes, followed by 35 cycles of 94 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 60 seconds. This was carried out by incubating at 3 ° C. for 3 minutes.
プライマーセット３
　　　フォワードプライマー３　5’-AGGAAACGAAGAATAGACG-3’　 （配列番号１０）
　　　リバースプライマー３　　5’-TGAGAACCGGAAAGAGAAGC-3’　（配列番号１１）
プライマーセット４
　　　フォワードプライマー４　5’-GTTCGGATCGGAAAATTCAA-3’　（配列番号１２）
　　　リバースプライマー４　　5’-CCAAGCTTTCCGACATTCAT-3’　（配列番号１３） Primer set 3
Forward primer 3 5'-AGGAAACGAAGAATAGACG-3 '(SEQ ID NO: 10)
Reverse primer 3 5'-TGAGAACCGGAAAGAGAAGC-3 '(SEQ ID NO: 11)
Primer set 4
Forward primer 4 5'-GTTCGGATCGGAAAATTCAA-3 '(SEQ ID NO: 12)
Reverse primer 4 5'-CCAAGCTTTCCGACATTCAT-3 '(SEQ ID NO: 13)
　そして、前記Ｆ２分離集団の前記増幅断片の長さ、前記第１ＳＮＰセット、前記第２ＳＮＰセット、および前記第３ＳＮＰセットについて、遺伝子型を分類した。また、前記Ｆ２分離集団について、前記（２）と同様にして、うどんこ病菌の接種試験を行い、発病指数を評価した。得られた結果の内、前記増幅断片の長さ、前記第１ＳＮＰセット、前記第２ＳＮＰセット、および前記第３ＳＮＰセットの遺伝子型が互いに異なる４個体（系統Ｘ１～Ｘ４）、前記寄託系統、前記罹病性系統、および前記寄託系統と前記罹病性系統とのＦ１系統の結果を表４に示す。なお、下記表４の増幅断片の長さにおいて、Ａは、２本の第６染色体から前記プライマーセット１で増幅される増幅断片の長さが共に１３３塩基以上であることを示し、Ｈは、１本の第６染色体から前記プライマーセット１で増幅される増幅断片の長さが１３３塩基以上であり、他方の染色体から前記プライマーセット１で増幅される増幅断片の長さが１３３塩基未満であることを示し、Ｂは、２本の第６染色体から増幅される前記増幅断片の長さが共に１３３塩基未満であることを示す。また、下記表４の第１ＳＮＰセット、第２ＳＮＰセット、および第３ＳＮＰセットにおいて、Ａは、各ＳＮＰセットが抵抗性のホモ接合型であることを示し、Ｈは、各ＳＮＰセットがヘテロ接合型であることを示し、Ｈは、各ＳＮＰセットが罹病性のホモ接合型であることを示す。下記表４において、Ａは網掛けで示している。 Then, genotypes were classified for the length of the amplified fragment of the F2 segregated population, the first SNP set, the second SNP set, and the third SNP set. Moreover, about the said F2 isolation | separation group, it carried out similarly to said (2), the powdery mildew inoculation test was done, and the disease onset index was evaluated. Among the obtained results, four individuals (strains X1 to X4) having different lengths of the amplified fragments, genotypes of the first SNP set, the second SNP set, and the third SNP set, the deposited strain, and the diseased disease Table 4 shows the results of the sex lines and the F1 lines of the deposited line and the susceptible line. In the lengths of the amplified fragments in Table 4 below, A indicates that the lengths of the amplified fragments amplified from the two chromosomes 6 by the primer set 1 are both 133 bases or more, and H is: The length of the amplified fragment amplified from one chromosome 6 with the primer set 1 is 133 bases or more, and the length of the amplified fragment amplified from the other chromosome with the primer set 1 is less than 133 bases And B indicates that the lengths of the amplified fragments amplified from two chromosomes 6 are both less than 133 bases. In the first SNP set, the second SNP set, and the third SNP set in Table 4 below, A indicates that each SNP set is a resistant homozygous type, and H indicates that each SNP set is a heterozygous type. H, indicating that each SNP set is susceptible to homozygosity. In Table 4 below, A is shaded.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　前記表４に示すように、前記増幅断片の長さおよび前記第１ＳＮＰセットが、Ａである個体においては、いずれの個体も発病指数が、１以下であった。これらの結果から、前記抵抗性遺伝子座において、前記増幅断片の長さおよび前記第１ＳＮＰセットが、前記うどんこ病抵抗性と高い相関を示すこと、すなわち、前記条件（１）および（２）が、前記うどんこ病抵抗性と高い相関を示す条件であることがわかった。また、前記増幅断片の長さおよび前記第１ＳＮＰセットと高い相関性を示すことから、これらを含む領域である前記第２ＳＮＰセットおよび前記第３ＳＮＰセットのいずれかのＳＮＰの部位間の領域が、前記うどんこ病抵抗性と高い相関を示すこと、すなわち、前記条件（９）が、前記うどんこ病抵抗性と高い相関を示す条件であることがわかった。これらの結果から、前記条件（１）および（２）の少なくとも一方を満たす抵抗性遺伝子座、ならびに前記条件（９）を満たす抵抗性遺伝子座が、メロン植物のうどんこ病抵抗性マーカーとなること、ならびに前記メロン植物のうどんこ病抵抗性マーカーを使用し、うどんこ病抵抗性メロン植物をスクリーニングできることがわかった。 As shown in Table 4, in the individuals in which the length of the amplified fragment and the first SNP set were A, the disease index of each individual was 1 or less. From these results, the length of the amplified fragment and the first SNP set show high correlation with the powdery mildew resistance at the resistance locus, that is, the conditions (1) and (2) The condition was highly correlated with the powdery mildew resistance. In addition, since it shows a high correlation with the length of the amplified fragment and the first SNP set, the region between the SNP sites of the second SNP set and the third SNP set, which are regions including these, It was found that there was a high correlation with powdery mildew resistance, that is, the condition (9) was a condition highly correlated with the powdery mildew resistance. From these results, the resistance locus satisfying at least one of the conditions (1) and (2) and the resistance locus satisfying the condition (9) become a powdery mildew resistance marker of melon plants. It was also found that the powdery mildew resistant melon plant can be screened using the powdery mildew resistance marker of the melon plant.
　以上、実施形態および実施例を参照して本発明を説明したが、本発明は、上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解し得る様々な変更をすることができる。 As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.
　この出願は、２０１５年１１月１３日に出願された日本出願特願２０１５－２２２７９４を基礎とする優先権を主張し、その開示の全てをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2015-222794 filed on November 13, 2015, the entire disclosure of which is incorporated herein.
　本発明のメロン植物のうどんこ病抵抗性マーカーによれば、例えば、うどんこ病抵抗性メロン植物を簡便にスクリーニングできる。また、本発明のうどんこ病抵抗性メロン植物は、例えば、前記うどんこ病抵抗性遺伝子座を含むため、例えば、うどんこ病抵抗性を示すことが可能である。さらに、前記うどんこ病抵抗性遺伝子座は、例えば、単一の遺伝子座でうどんこ病抵抗性を付与できる。このため、本発明のうどんこ病抵抗性メロン植物は、例えば、他のメロン植物との交雑によって得られたＦ１またはその後代からも、うどんこ病抵抗性を示す後代を簡便に得ることができる。さらに、前記うどんこ病抵抗性マーカーを含むメロン植物は、例えば、前記先行技術文献のうどんこ病抵抗性遺伝子を含むメロン植物に対して感染可能なうどんこ病菌に対しても抵抗性を示す。このため、本発明のうどんこ病抵抗性メロン植物は、従来のような農薬による防除が不要であるため、例えば、前記農薬散布の労力および費用の問題も回避できる。 According to the powdery mildew resistance marker for melon plants of the present invention, for example, powdery mildew resistant melon plants can be easily screened. Moreover, since the powdery mildew resistant melon plant of this invention contains the said powdery mildew resistant gene locus, for example, it can show powdery mildew resistant, for example. Furthermore, the powdery mildew resistance locus can confer powdery mildew resistance at, for example, a single locus. For this reason, the powdery mildew resistant melon plant of this invention can obtain easily the progeny which shows powdery mildew resistance from F1 obtained by crossing with another melon plant, or its progeny, for example. . Furthermore, the melon plant containing the powdery mildew resistance marker also exhibits resistance to powdery mildew bacteria that can infect melon plants containing the powdery mildew resistance gene of the prior art document, for example. For this reason, since the powdery mildew resistant melon plant of this invention does not need the control by the conventional agrochemical, the problem of the labor and cost of the said agrochemical spraying can also be avoided, for example.

Claims (20)

  1. 第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする、メロン植物のうどんこ病抵抗性マーカー。
    条件（１）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
    前記増幅断片の長さが、１３３塩基長以上である。
    プライマーセット１
    　　　配列番号１の塩基配列からなるフォワードプライマー１
    　　　配列番号２の塩基配列からなるリバースプライマー１
    条件（２）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。     Including a powdery mildew resistance locus on chromosome 6 in homozygous form,
    A powdery mildew resistance marker for melon plants, wherein the powdery mildew resistance locus on chromosome 6 satisfies at least one of the following conditions (1) and (2):
    Condition (1)
    The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
    The length of the amplified fragment is 133 bases or more.
    Primer set 1
    Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
    Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
    Condition (2)
    The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  2. 前記条件（２）において、前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列により特定される、請求項１記載のメロン植物のうどんこ病抵抗性マーカー。 The melon plant according to claim 1, wherein, in the condition (2), the powdery mildew resistance locus on the chromosome 6 is specified by the 204th to 212th and 213th base sequences in the base sequence of SEQ ID NO: 3. Powdery mildew resistance marker.
  3. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（３）および（４）の少なくとも一方の条件を満たす、請求項１または２記載のメロン植物のうどんこ病抵抗性マーカー。
    条件（３）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号４の塩基配列における５０、１４１、および２６６番目の塩基の多型で特定される。
    条件（４）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号５の塩基配列における９９、１７４、１８４、１９９、および２００番目の塩基の多型で特定される。     The powdery mildew resistance marker for melon plants according to claim 1 or 2, wherein the powdery mildew resistance locus on chromosome 6 satisfies at least one of the following conditions (3) and (4).
    Condition (3)
    The powdery mildew resistance locus on the sixth chromosome is specified by polymorphisms of the 50th, 141st and 266th bases in the base sequence of SEQ ID NO: 4.
    Condition (4)
    The powdery mildew resistance locus on the sixth chromosome is identified by polymorphisms of the 99th, 174th, 184th, 199th and 200th bases in the base sequence of SEQ ID NO: 5.
  4. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（５）および（６）の少なくとも一方の条件を満たす、請求項１から３のいずれか一項に記載のメロン植物のうどんこ病抵抗性マーカー。
    条件（５）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ａ）のポリヌクレオチドで特定される。
    （ａ）　下記（ａ１）のポリヌクレオチド
    （ａ１）配列番号６の塩基配列からなるポリヌクレオチド
    条件（６）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｂ）のポリヌクレオチドで特定される。
    （ｂ）　下記（ｂ１）のポリヌクレオチド
    （ｂ１）配列番号３の塩基配列からなるポリヌクレオチド     4. The powdery mildew of the melon plant according to claim 1, wherein the powdery mildew resistance locus on the chromosome 6 satisfies at least one of the following conditions (5) and (6): Disease resistance marker.
    Condition (5)
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (a) below.
    (A) Polynucleotide condition (6) consisting of the polynucleotide of (a1) below (a1) SEQ ID NO: 6
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (b) below.
    (B) a polynucleotide of the following (b1) (b1) a polynucleotide comprising the base sequence of SEQ ID NO: 3
  5. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（７）および（８）の少なくとも一方の条件を満たす、請求項１から４のいずれか一項に記載のメロン植物のうどんこ病抵抗性マーカー。
    条件（７）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｃ）のポリヌクレオチドで特定される。
    （ｃ）　下記（ｃ１）のポリヌクレオチド
    （ｃ１）配列番号４の塩基配列からなるポリヌクレオチド
    条件（８）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｄ）のポリヌクレオチドで特定される。
    （ｄ）　下記（ｄ１）のポリヌクレオチド
    （ｄ１）配列番号５の塩基配列からなるポリヌクレオチド     The powdery mildew resistant locus on the sixth chromosome of the melon plant according to any one of claims 1 to 4, wherein at least one of the following conditions (7) and (8) is satisfied: Disease resistance marker.
    Condition (7)
    The powdery mildew resistance locus on chromosome 6 is specified by the polynucleotide (c) below.
    (C) Polynucleotide condition (8) comprising the nucleotide sequence of SEQ ID NO: 4 (c1) polynucleotide (c1) below
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (d) below.
    (D) a polynucleotide of the following (d1) (d1) a polynucleotide comprising the base sequence of SEQ ID NO: 5
  6. 第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含み、
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たすことを特徴とする、うどんこ病抵抗性メロン植物。
    条件（１）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
    前記増幅断片の長さが、１３３塩基長以上である。
    プライマーセット１
    　　　配列番号１の塩基配列からなるフォワードプライマー１
    　　　配列番号２の塩基配列からなるリバースプライマー１
    条件（２）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。     Including a powdery mildew resistance locus on chromosome 6 in homozygous form,
    The powdery mildew resistant melon plant, wherein the powdery mildew resistant locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2):
    Condition (1)
    The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
    The length of the amplified fragment is 133 bases or more.
    Primer set 1
    Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
    Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
    Condition (2)
    The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  7. 前記条件（２）において、前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列により特定される、請求項６記載のうどんこ病抵抗性メロン植物。 7. The powdery mildew according to claim 6, wherein the powdery mildew resistance locus on the chromosome 6 is specified by the 204-212 and 213th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 3 in the condition (2). Disease-resistant melon plant.
  8. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（３）および（４）の少なくとも一方の条件を満たす、請求項６または７記載のうどんこ病抵抗性メロン植物。
    条件（３）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号４の塩基配列における５０、１４１、および２６６番目の塩基の多型で特定される。
    条件（４）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号５の塩基配列における９９、１７４、１８４、１９９、および２００番目の塩基の多型で特定される。     The powdery mildew resistant melon plant according to claim 6 or 7, wherein the powdery mildew resistant locus on the sixth chromosome satisfies at least one of the following conditions (3) and (4).
    Condition (3)
    The powdery mildew resistance locus on the sixth chromosome is specified by polymorphisms of the 50th, 141st and 266th bases in the base sequence of SEQ ID NO: 4.
    Condition (4)
    The powdery mildew resistance locus on the sixth chromosome is identified by polymorphisms of the 99th, 174th, 184th, 199th and 200th bases in the base sequence of SEQ ID NO: 5.
  9. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（５）および（６）の少なくとも一方の条件を満たす、請求項６から８のいずれか一項に記載のうどんこ病抵抗性メロン植物。
    条件（５）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ａ）のポリヌクレオチドで特定される。
    （ａ）　下記（ａ１）のポリヌクレオチド
    （ａ１）配列番号６の塩基配列からなるポリヌクレオチド
    条件（６）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｂ）のポリヌクレオチドで特定される。
    （ｂ）　下記（ｂ１）のポリヌクレオチド
    （ｂ１）配列番号３の塩基配列からなるポリヌクレオチド     The powdery mildew resistance locus according to any one of claims 6 to 8, wherein the powdery mildew resistance locus on the chromosome 6 satisfies at least one of the following conditions (5) and (6): Melon plant.
    Condition (5)
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (a) below.
    (A) Polynucleotide condition (6) consisting of the polynucleotide of (a1) below (a1) SEQ ID NO: 6
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (b) below.
    (B) a polynucleotide of the following (b1) (b1) a polynucleotide comprising the base sequence of SEQ ID NO: 3
  10. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（７）および（８）の少なくとも一方の条件を満たす、請求項６から９のいずれか一項に記載のうどんこ病抵抗性メロン植物。
    条件（７）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｃ）のポリヌクレオチドで特定される。
    （ｃ）　下記（ｃ１）のポリヌクレオチド
    （ｃ１）配列番号４の塩基配列からなるポリヌクレオチド
    条件（８）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｄ）のポリヌクレオチドで特定される。
    （ｄ）　下記（ｄ１）のポリヌクレオチド
    （ｄ１）配列番号５の塩基配列からなるポリヌクレオチド     Powdery mildew resistance according to any one of claims 6 to 9, wherein the powdery mildew resistance locus on the chromosome 6 satisfies at least one of the following conditions (7) and (8): Melon plant.
    Condition (7)
    The powdery mildew resistance locus on chromosome 6 is specified by the polynucleotide (c) below.
    (C) Polynucleotide condition (8) comprising the nucleotide sequence of SEQ ID NO: 4 (c1) polynucleotide (c1) below
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (d) below.
    (D) a polynucleotide of the following (d1) (d1) a polynucleotide comprising the base sequence of SEQ ID NO: 5
  11. 前記うどんこ病抵抗性メロン植物が、受託番号ＦＥＲＭ　ＢＰ－２２２９１で特定されるメロン植物またはその後代系統である、請求項６から１０のいずれか一項に記載のうどんこ病抵抗性メロン植物。 The powdery mildew resistant melon plant according to any one of claims 6 to 10, wherein the powdery mildew resistant melon plant is a melon plant identified by the accession number FERM BP-22291 or a progeny line thereof.
  12. 前記うどんこ病抵抗性メロン植物が、植物体またはその部分である、請求項６から１１のいずれか一項に記載のうどんこ病抵抗性メロン植物。 The powdery mildew resistant melon plant according to any one of claims 6 to 11, wherein the powdery mildew resistant melon plant is a plant body or a part thereof.
  13. 前記うどんこ病抵抗性メロン植物が、種子である、請求項６から１２のいずれか一項に記載のうどんこ病抵抗性メロン植物。 The powdery mildew resistant melon plant according to any one of claims 6 to 12, wherein the powdery mildew resistant melon plant is a seed.
  14. 下記（ａ）および（ｂ）工程を含むことを特徴とする、うどんこ病抵抗性メロン植物の製造方法。
    （ａ）請求項６から１３のいずれか一項に記載のうどんこ病抵抗性メロン植物と、他のメロン植物とを交雑する工程
    （ｂ）前記（ａ）工程より得られたメロン植物またはその後代系統から、うどんこ病抵抗性メロン植物を選抜する工程     A method for producing a powdery mildew resistant melon plant comprising the following steps (a) and (b):
    (A) a step of crossing the powdery mildew resistant melon plant according to any one of claims 6 to 13 with another melon plant (b) a melon plant obtained from the step (a) or thereafter Process for selecting powdery mildew resistant melon plants from different strains
  15. 前記（ａ）工程に先立って、下記（ｘ）工程を含む、請求項１４記載のうどんこ病抵抗性メロン植物の製造方法。
    （ｘ）被検メロン植物から、請求項６から１３のいずれか一項に記載のうどんこ病抵抗性メロン植物を選抜する工程     The method for producing a powdery mildew resistant melon plant according to claim 14, comprising the following step (x) prior to the step (a).
    (X) A step of selecting the powdery mildew resistant melon plant according to any one of claims 6 to 13 from the test melon plant.
  16. 前記（ｘ）工程における前記選抜が、第６染色体上のうどんこ病抵抗性遺伝子座をホモ接合型で含むうどんこ病抵抗性メロン植物の選抜であり、
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（１）および（２）の少なくとも一方の条件を満たす、請求項１５記載のうどんこ病抵抗性メロン植物の製造方法。
    条件（１）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記プライマーセット１で増幅される増幅断片の長さで特定され、
    前記増幅断片の長さが、１３３塩基長以上である。
    プライマーセット１
    　　　配列番号１の塩基配列からなるフォワードプライマー１
    　　　配列番号２の塩基配列からなるリバースプライマー１
    条件（２）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における４５、４８、４９、５１、１０８、１２０、１３９、２１４、および３２７番目の塩基の多型で特定される。     The selection in the step (x) is selection of a powdery mildew resistant melon plant containing a powdery mildew resistant locus on chromosome 6 in a homozygous form,
    The method for producing a powdery mildew resistant melon plant according to claim 15, wherein the powdery mildew resistant locus on the sixth chromosome satisfies at least one of the following conditions (1) and (2).
    Condition (1)
    The powdery mildew resistance locus on the chromosome 6 is identified by the length of the amplified fragment amplified by the following primer set 1,
    The length of the amplified fragment is 133 bases or more.
    Primer set 1
    Forward primer 1 consisting of the base sequence of SEQ ID NO: 1
    Reverse primer 1 consisting of the base sequence of SEQ ID NO: 2
    Condition (2)
    The powdery mildew resistance locus on the sixth chromosome is specified by the polymorphisms of the 45th, 48th, 49th, 51th, 108th, 120th, 139th, 214th, and 327th bases in the base sequence of SEQ ID NO: 3. .
  17. 前記条件（２）において、前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号３の塩基配列における２０４～２１２および２１３番目の塩基配列により特定される、請求項１６記載のうどんこ病抵抗性メロン植物の製造方法。 The powdery mildew resistance locus on the chromosome 6 is identified by the 204-212 and 213th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 3 in the condition (2), A method for producing a disease-resistant melon plant.
  18. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（３）および（４）の少なくとも一方の条件を満たす、請求項１６または１７記載のうどんこ病抵抗性メロン植物の製造方法。
    条件（３）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号４の塩基配列における５０、１４１、および２６６番目の塩基の多型で特定される。
    条件（４）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、配列番号５の塩基配列における９９、１７４、１８４、１９９、および２００番目の塩基の多型で特定される。     The method for producing a powdery mildew resistant melon plant according to claim 16 or 17, wherein the powdery mildew resistant locus on the sixth chromosome satisfies at least one of the following conditions (3) and (4).
    Condition (3)
    The powdery mildew resistance locus on the sixth chromosome is specified by polymorphisms of the 50th, 141st and 266th bases in the base sequence of SEQ ID NO: 4.
    Condition (4)
    The powdery mildew resistance locus on the sixth chromosome is identified by polymorphisms of the 99th, 174th, 184th, 199th and 200th bases in the base sequence of SEQ ID NO: 5.
  19. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（５）および（６）の少なくとも一方の条件を満たす、請求項１６から１８のいずれか一項に記載のうどんこ病抵抗性メロン植物の製造方法。
    条件（５）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ａ）のポリヌクレオチドで特定される。
    （ａ）　下記（ａ１）のポリヌクレオチド
    （ａ１）配列番号６の塩基配列からなるポリヌクレオチド
    条件（６）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｂ）のポリヌクレオチドで特定される。
    （ｂ）　下記（ｂ１）のポリヌクレオチド
    （ｂ１）配列番号３の塩基配列からなるポリヌクレオチド     The powdery mildew resistance locus according to any one of claims 16 to 18, wherein the powdery mildew resistance locus on the chromosome 6 satisfies at least one of the following conditions (5) and (6): Method for producing melon plant.
    Condition (5)
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (a) below.
    (A) Polynucleotide condition (6) consisting of the polynucleotide of (a1) below (a1) SEQ ID NO: 6
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (b) below.
    (B) a polynucleotide of the following (b1) (b1) a polynucleotide comprising the base sequence of SEQ ID NO: 3
  20. 前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記条件（７）および（８）の少なくとも一方の条件を満たす、請求項１６から１９のいずれか一項に記載のうどんこ病抵抗性メロン植物の製造方法。
    条件（７）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｃ）のポリヌクレオチドで特定される。
    （ｃ）　下記（ｃ１）のポリヌクレオチド
    （ｃ１）配列番号４の塩基配列からなるポリヌクレオチド
    条件（８）
    前記第６染色体上のうどんこ病抵抗性遺伝子座が、下記（ｄ）のポリヌクレオチドで特定される。
    （ｄ）　下記（ｄ１）のポリヌクレオチド
    （ｄ１）配列番号５の塩基配列からなるポリヌクレオチド     The powdery mildew resistance locus according to any one of claims 16 to 19, wherein the powdery mildew resistance locus on the sixth chromosome satisfies at least one of the following conditions (7) and (8): Method for producing melon plant.
    Condition (7)
    The powdery mildew resistance locus on chromosome 6 is specified by the polynucleotide (c) below.
    (C) Polynucleotide condition (8) comprising the nucleotide sequence of SEQ ID NO: 4 (c1) polynucleotide (c1) below
    The powdery mildew resistance locus on the sixth chromosome is specified by the polynucleotide (d) below.
    (D) a polynucleotide of the following (d1) (d1) a polynucleotide comprising the base sequence of SEQ ID NO: 5
PCT/JP2016/066009 2015-11-13 2016-05-31 White mildew resistance marker for melon plants, white mildew-resistant melon plant, and method for producing white mildew-resistant melon plant using said white mildew-resistant melon plant WO2017081876A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
TR2018/06450T TR201806450T1 (en) 2015-11-13 2016-05-31 Powdery mildew resistance marker for melon plants, powdery mildew resistant melon plant and method for producing powdery mildew resistant melon plant using them.
ES201890033A ES2691593B1 (en) 2015-11-13 2016-05-31 OID RESISTANCE MARKER FOR MELON PLANTS, OIDIO RESISTANT MELON PLANT, AND METHOD FOR PRODUCING OIDIO RESISTANT MELON PLANT USING SUCH MARKER
US15/775,637 US10772273B2 (en) 2015-11-13 2016-05-31 Powdery mildew resistance marker for melon plants, powdery mildew resistant melon plant, and method for producing powdery mildew resistant melon plant using the same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2015222794A JP6811526B2 (en) 2015-11-13 2015-11-13 Powdery mildew resistance marker of melon plant, powdery mildew resistant melon plant, and method for producing powdery mildew resistant melon plant using it.
JP2015-222794 2015-11-13

Publications (1)

Publication Number Publication Date
WO2017081876A1 true WO2017081876A1 (en) 2017-05-18

Family

ID=58694949

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2016/066009 WO2017081876A1 (en) 2015-11-13 2016-05-31 White mildew resistance marker for melon plants, white mildew-resistant melon plant, and method for producing white mildew-resistant melon plant using said white mildew-resistant melon plant

Country Status (5)

Country Link
US (1) US10772273B2 (en)
JP (1) JP6811526B2 (en)
ES (1) ES2691593B1 (en)
TR (1) TR201806450T1 (en)
WO (1) WO2017081876A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11519003B2 (en) 2018-03-30 2022-12-06 Keygene N.V. QTLs for powdery mildew resistance in melon

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6306252B1 (en) * 2017-07-31 2018-04-04 タキイ種苗株式会社 Powdery mildew resistance marker of pumpkin plant, powdery mildew resistant pumpkin plant, method for producing powdery mildew resistant pumpkin plant using the same, and method for imparting powdery mildew resistance to pumpkin plant
WO2019174721A1 (en) * 2018-03-13 2019-09-19 Dümmen Group B.V. Tobamovirus resistant solanaceae plant
CN111121629B (en) * 2019-12-31 2021-03-26 广西壮族自治区农业科学院 Soybean leaf wrinkling field identification method
KR102369882B1 (en) * 2020-09-29 2022-03-02 세종대학교산학협력단 Molecular marker for identification of melon resistant to powdery mildew and identification method using the same marker

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FUKINO N. ET AL.: "Identification of QTLs for resistance to powdery mildew and SSR markers diagnostic for powdery mildew resistance genes in melon (Cucumis melo L.).", THEOR. APPL. GENET., vol. 118, no. 1, 2008, pages 165 - 175, XP019651278 *
MAKI KUZUYA ET AL.: "Meron Udonkobyokin no Race Bunka to Teikosei Ikushu", PROCEEDINGS OF VEGETABLE AND TEA SCIENCE, 2004, pages 39 - 43 *
PERCHEPIED L. ET AL.: "Relationship Between Loci Conferring Downy Mildew and Powdery Mildew Resistance in Melon Assessed by Quantitative Trait Loci Mapping.", PHYTOPATHOLOGY, vol. 95, no. 5, 2005, pages 556 - 565, XP055022414 *
YUSTE-LISBONA F.J. ET AL.: "Codominant PCR-based markers and candidate genes for powdery mildew resistance in melon (Cucumis melo L.).", THEOR. APPL. GENET., vol. 122, no. 4, 2011, pages 747 - 758, XP019881302 *
YUSTE-LISBONA F.J. ET AL.: "Genetic linkage map of melon (Cucumis melo L.) and localization of a major QTL for powdery mildew resistance.", MOL. BREEDING, vol. 27, no. 2, 2011, pages 181 - 192, XP019880334 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11519003B2 (en) 2018-03-30 2022-12-06 Keygene N.V. QTLs for powdery mildew resistance in melon
US11950554B2 (en) 2018-03-30 2024-04-09 Keygene N.V. QTLS for powdery mildew resistance in melon

Also Published As

Publication number Publication date
ES2691593B1 (en) 2019-09-13
JP6811526B2 (en) 2021-01-13
ES2691593R1 (en) 2019-01-22
TR201806450T1 (en) 2018-06-21
US20180325057A1 (en) 2018-11-15
ES2691593A2 (en) 2018-11-27
US10772273B2 (en) 2020-09-15
JP2017086016A (en) 2017-05-25

Similar Documents

Publication Publication Date Title
EP2219433B1 (en) New cucumber plants with a compact growing habit
JP6306252B1 (en) Powdery mildew resistance marker of pumpkin plant, powdery mildew resistant pumpkin plant, method for producing powdery mildew resistant pumpkin plant using the same, and method for imparting powdery mildew resistance to pumpkin plant
WO2017081876A1 (en) White mildew resistance marker for melon plants, white mildew-resistant melon plant, and method for producing white mildew-resistant melon plant using said white mildew-resistant melon plant
JP6412497B2 (en) Root-knot nematode resistance marker for tomato plant, root-knot nematode-resistant tomato plant, method for producing root-knot nematode-resistant tomato plant, and method for screening root-knot nematode-resistant tomato plant
NL2002357C2 (en) Methods for improving the yield of cucumber plants.
WO2008003783A2 (en) Resistance to powdery mildew and absence of necrosis in cucumis sativus
JP6936626B2 (en) Half-body wilt resistance marker of tomato plant, half-body wilt-resistant tomato plant, and method for producing half-body wilt-resistant tomato plant using the same.
KR102334176B1 (en) Leaf mold disease resistance marker of tomato plant, foliar mold disease resistant tomato plant, method for producing foliar mold disease resistant tomato plant and screening method for foliar mold disease resistant tomato plant
JP6299030B2 (en) PRSV-resistant cucumber plant and method for producing cucumber plant exhibiting PRSV resistance and ZYMV resistance
JP7357173B1 (en) sooty mold resistant tomato plants
WO2023068371A1 (en) Eustoma plant having eustoma dieback resistance
JP7239790B1 (en) Eustoma Take-all Resistant Eustoma Plants
JP2019024483A (en) Mildew resistant pumpkin plants using the same, and methods for imparting powdery mildew resistance to pumpkin plants
JP2023049045A (en) Phomopsis root rot disease-resistant pumpkin plant
JP6153245B2 (en) Brown spot disease-resistant cucumber plant, and method for producing cucumber plant exhibiting brown spot resistance and powdery mildew resistance
WO2024003885A1 (en) Novel markers combination in sweet basil for disease resistances
EP2041289A2 (en) Resistance to powdery mildew and absence of necrosis in cucumis sativus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16863840

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: P201890033

Country of ref document: ES

WWE Wipo information: entry into national phase

Ref document number: 15775637

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16863840

Country of ref document: EP

Kind code of ref document: A1